Dissertations / Theses on the topic 'Recombinant protein'

Orientador: Nilson Ivo Tonin Zanchin
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A quinase de 70 kDa da proteína ribossomal S6, isoforma 1 (S6K1), é uma fosfoproteína implicada na regulação de genes relacionados ao controle da tradução em mamíferos e possui uma forma nuclear (a1) e uma citoplasmática (a2). A fosforilação do seu principal alvo, a proteína RPS6, tem sido comumente associada ao recrutamento seletivo dos 5'-TOP (5' tract of oligopyrimidine) mRNAs pela maquinaria de tradução, embora haja estudos contrariando esta hipótese. Devido às funções de seus demais alvos, S6K1 tem sido implicada na sobrevivência celular e em diversos outros processos, como crescimento, câncer e resistência à insulina. S6K1 é ativada por um mecanismo que envolve fosforilação seqüencial através da ativação das vias mTORC1 (complexo 1 do alvo da rapamicina em mamíferos) e PI3K (fosfoinositol-3 quinase). Como uma quinase da família AGC, S6K1 deve ser fosforilada por mTORC1 no resíduo Thr389 do domínio hidrofóbico e, em seguida, por PDPK1 (proteína quinase 1 dependente de fosfoinositol) no resíduo Thr229 da alça T do domínio catalítico. Estes eventos ocorrem somente após a fosforilação em diversos sítios do domínio auto-inibitório carboxiterminal, por mTORC1. O objetivo deste trabalho foi desenvolver um ensaio modelo para análise da função da S6K1 in vitro e utilizá-lo como ferramenta na elucidação do papel de proteínas adaptadoras da via de mTOR em interações com a S6K1. Para isso foi necessário produzir as proteínas recombinantes para ensaios de interação e para realização de um ensaio de atividade para a S6K1. Foram testados vários sistemas de expressão para Escherichia coli para produção das construções GST-S6K1a1-His6, GST-S6K1a2-His6 e GST-S6K1a2T389E?CT (forma a2 de S6K1 com a substituição T389E e o carboxiterminal truncado), GST-PDPK1 e GST-CDPDPK1 (domínio catalítico de PDPK1 fusionado a GST). A expressão das formas truncadas de S6K1 e PDPK1 foi mais eficiente em E. coli. Embora o rendimento tenha ficado muito aquém do esperado, foi suficiente para os ensaios de interação in vitro. Também foi feita a expressão em E. coli da região C-terminal da proteína RPS6, que é o substrato da S6K1, em fusão com a proteína D do fago ?. Posteriormente, foram montados sistemas de expressão das construções His6-S6K1a2T389E?CT e His6-CDPDPK1 em células de inseto, a partir de vetor de baculovírus. Constatou-se que essas construções são expressas na forma de fosfoproteínas em células de inseto. Ensaios de GST pull-down com GST-S6K1a2-His6 e GST-S6K1a2T389E?CT contra as duas isoformas da subunidade catalítica da PP2AC, His6-PP2ACa(maior) e His6-PP2ACa(menor), revelaram que His6-PP2ACa(maior) não interage com GST-S6K1a2-His6, embora interaja fortemente com GST-S6K1a2T389E?CT. Já a construção His6-PP2ACa(menor) interage fracamente com as construções GST-S6K1a2-His6 e GST-S6K1a2T389E?CT. Tomados em conjunto, os resultados sugerem que a presença do C-terminal não fosforilado de S6K1a2 impede a interação com PP2ACa(maior). PP2ACa(menor) comporta-se de forma completamente diferente da isoforma maior, pois a interação entre PP2ACa(menor) e S6K1a2 parece ser independente do carboxiterminal da quinase, visto que as quantidades de S6K1a2T389E?CT e de S6K1a2 inteira que interagem com PP2ACa(menor) são semelhantes. Esses resultados necessitam ainda serem confirmados in vivo. Outros experimentos de GST pull-down confirmaram que as construções de S6K1 não interagem com a4, embora interajam com TIPRL1. Se confirmado in vivo, esse resultado compõe um novo quadro na regulação coordenada entre mTOR1 e PP2A, do qual TIPRL1 parece participar. As construções genéticas e os sistemas de expressão gerados neste trabalho possibilitaram a obtenção dos reagentes necessários para analisar o mecanismo de regulação da quinase S6K1, mediado por proteínas regulatórias. Permitem também desenvolver uma série de experimentos, como busca de inibidores específicos para a S6K1, que dependem da reconstituição de ensaios de atividade in vitro com a S6K1 ativada. Contudo, o ensaio de atividade realizado não apresentou resultados satisfatórios e precisa ser desenvolvido.
Abstract: The 70kDa ribosomal S6 protein kinase 1 (S6K1) is a phosphoprotein involved in the regulation of genes related to translational control in mammals. S6K1 shows distinct nuclear (a1) and cytoplasmic (a2) forms. Phosphorylation of the S6K1 best characterized target, the protein of the small ribosomal subunit (RPS6), has been generally associated to the selective recruitment of the 5'-TOP mRNAs (5' tract of oligopyrimidine) by the translational machinery, although there is still some controversy on this issue. Due to the function of its targets, S6K1 has been implicated in several cellular processes including cell growth, cancer and insulin resistance. S6K1 is activated by a mechanism of sequential phosphorylation following activation of the mTORC1 (mammalian target of rapamycin complex 1) and PI3K (phosphoinositide-3-kinase) pathways. As a kinase of the AGC family, S6K1 activation requires mTORC1 phosphorylation of residue Thr389 of the hydrophobic domain followed by PDPK1 (phosphoinositide dependent protein kinase 1) phosphorylation of residue Thr229 at the T loop of the catalytic domain. These take place only after phosphorylation by mTORC1 of several residues of the autoinhibitory C-terminal domain. The objective of this work was to develop an assay to analyze the function of S6K1 in vitro and use it as a tool in the discovering of the functions of regulators proteins of the mTOR cascade in interactions with S6K1. For these purposes, expression systems were constructed to produce the various recombinant proteins to be used in the interaction and activity assays. Several genetic constructions were tested in Escherichia coli for the production of GST-S6K1a1-His6, GST-S6K1a2-His6 and GST-S6K1a2T389E?CT (a2 form of S6K1 with the T389E substitution and truncated carboxiterminus), GST-PDPK1 and GST-CDPDPK1 (GST fusion protein of the catalytic domain of PDPK1). The truncated forms were expressed more efficiently in E. coli. Although the yield in E. coli was lower than expected, it was sufficient to perform interaction assays. The C-terminal domain of RPS6, a substrate for S6K1, was successfully expressed in E. coli as a fusion protein with the phage ? protein D. Subsequently, expression systems for production of His6-S6K1a2T389E?CT and His6-CDPDPK1 in insect cells were constructed using baculovirus vectors. It was found that these constructs are expressed in the form of phosphoproteins in insect cells. GST pull-down assays using GST-S6K1a2-His6 e GST-S6K1a2T389E?CT to test interaction with the PP2AC isoforms His6-PP2ACa(major) and His6-PP2ACa(minor) revealed that His6-PP2ACa(major) does not interact with GST-S6K1a2-His6, although it interacts strongly with GST-S6K1a2T389E?CT. On the other hand, His6-PP2ACa(minor) interacts weakly with both GST- S6K1a2-His6 and GST-S6K1a2T389E?CT. This finding suggests that the unphosphorylated C-terminal of S6K1a2 inhibits interaction with PP2ACa(major). His6-PP2ACa(minor) behaves differently form His6-PP2ACa(major). Its interaction with S6K1a2 seems to be independent of the C-terminal since the amounts of S6K1a2T389E?CT and S6K1a2 that interact with His6-PP2ACa(minor) are similar. Future work in vivo is required to confirm these results. GST pull-down assays confirmed that a4 does not interact with the constructions of S6K1, while TIPRL1 interacts with them. If confirmed in vivo, these results provides a new perspective for the coordinated regulation between mTOR1 and PP2A, which apparently involves also TIPRL1. The genetic constructions and expression systems established in this work allow the production of the reagents required to study the mechanism of S6K1 regulation mediated by adaptor proteins. They will also allow the development of experiments such as screening for specific S6K1 inhibitors, which depend on reconstitution of S6K1 activity assays using activated S6K1. Nevertheless, the activity assay performed did not yield satisfactory outcomes and must be improved.
Mestrado
Bioquimica
Mestre em Biologia Funcional e Molecular

No Brasil, e em outros paises de clima tropical, os carrapatos se tornaram um enorme problema economico de^$de que a industria do gado se desenvolveu. O carrapato Boophilus microplus, um dos artropodes mais importantes na veterinaria, causa efeitos direto, como suc,cao de sangue, e indireto, como a transmissao de uma grande variedade de patogenos que normalmente resulta em infec,c~es letais. As vacinas genicas contendo o antigeno Bm86, uma proteina ligada a membrana do intestino do carrapato B. microplus, representam uma alternativa atrativa aos acaricidas para controlar as infesta~oes por carrapatos em contrapartida aos inconvenientes produtos quimicos. Devido sua administra,cao ser feita em 4 doses no primeiro ano, seguida de refor,cos a cada seis meses, estas formula,coes vacinais nao s3c adequadas para paises com cria,cao extensiva de gado, como no Brasil. Visando uma libera~ao sustentada do antigeno Bm86, neste trabalho desenvolveuse uma vacina de dose unica baseada em microesferas polimericas. Para obter o padrao de liberac,ao desejavel, diferentes formula,coes e parametros de processo foram variados, como a composi,cao do polimero, a taxa entre os monomeros ^Uacido latico:acido glicolico\" e o tamanho das microparticulas. As formula,coes foram preparadas pelo metodo de emulsao multipla e evapora,cao do solvente. A formula~ao que melhor se enquadrou nos objetivos da vacina de dose unica foi preparada com PLGA 75:25, solu,cao 3% de PVA como estabilizante, agita,cao de 11000 rpm para forma,cao da emulsao primaria e de 800 rpm para forma,cao da emulsao multipla e evapora,cao do solvente. As particulas assim obtidas apresentaram um tamanho medio de 25 ,um, uma taxa de encapsula,cao maior que 90% e aproximadamente 50% da proteina foi liberada in vitro em 60 dias. Analises por SDS-PAGE e Westem Bloning revelaram que a proteina se manteve integra apos encapsula,cao. Os resultados da avalia,cao da imunogenicidade em bovinos mostraram que a formula,cao baseada em microesferas polimericas biodegradaveis e habil a conseguir, com uma unica dose, uma resposta imune similar aquela conseguida com tres doses das formula,coes convencionais da vacina de Bm86.
In Brazil, and in others tropical countries, the ticks have become a huge economic problem since the industry of livestock has developed. Ticks and tick-borne diseases affect animal and human health and are the cause of significant economic losses. The cattle tick Boophilus microplus is one of the most important arthropods in veterinary. This tick species causes both direct effects, such as blood sucking, and indirect effects, such as transmission of a wide variety of pathogens, which usually result in lethal infections. The gene vaccines based on Bm86 antigen, a midgut membrane-bound protein of the cattle tick B. microplus, represent a good alternative to control tick infestations, compared to chemicals. However, due to these vaccine formulations need 4 doses over the first year with booster at each 6 months to be effective, they are not suitable for countries with extensive cattle raising, like Brazil. Aiming a sustained release of Bm86 antigen, in this work we developed a single shot vaccine based on Bm86 loaded polymeric microspheres. In order to obtain desired release patterns, different formulations and processing parameters were varied, for example, the composition of the polymer, the monomer ratio lactic acid:glycolic acid and the size of the microparticles. The formulations were prepared by solvent evaporation method based on double emulsion. The formulation that presented better result as single shot vaccine was prepared with PLGA 75:25, solution 3% of PVA as stabilizer, agitation of 11000 rpm to form the primary emulsion and 800 rpm to obtain the double emulsion and solvent evaporation. The particles thus obtained presented an average size of 25 m, encapsulation ratio greater than 90% and approximately 50% of the protein was released in vitro in 60 days. Analysis by SDSPAGE and Western Blot showed that the integrity of the protein remained after encapsulation. The immunogenic studies showed that the formulation based onbiodegradable polymeric microspheres is able to elicit, with a single dose, an immune response and protection similar to that attained with 3 doses of conventional Bm86 vaccine formulations.

The aim of this thesis was to produce DNA expression constructs and use them to investigate the feasibility of recombinantly expression proteins for future interaction studies between human RBBP6 and p53 and pRb proteins.

Thesis (Ph. D.) -- University of Maryland, College Park, 2004.
Thesis research directed by: Chemical Engineering. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Degut a la seva flexibilitat funcional i arquitectònica, l’ús de proteïnes recombinants formades per múltiples dominis representen una eina interesant pel desenvolupament de nanopartícules capaces de transportar fàrmacs de manera específica. Recentment, en el nostre grup, hem aplicat un nou sistema d’enginyeria pel desenvolupament de nanoparticles formades mitjançant l’oligomerització de proteïnes modulars. L’eficàcia selectiva i l’absència de toxicitat d’unes nanopartícules dirigides al càncer colorectal demostra la capacitat d’aquest enfoc biotecnològic. Un enfoc basat en l’ús de pèptids catiònics en els extrems terminal d’una proteïna modular que li confereix la capacitat d’interaccionar amb sí mateixa formant nanopartícules proteiques. Per tal d’explorar la flexibilitat d’aquest enfoc, hem desenvolupat dues nanopartícules proteiques, anomenades A5G27-GFP-H6 i FNI/II/V-GFP-H6, que reconeixen i internalitzen, de manera específica, cèl·lules que expressen el receptor CD44. Aquestes proteïnes modulars formen estructures anulars de 14 nm, estables en plasma capaces d’internalitzar a través d’endocitosis sense presentar toxicitat cel·lular. Aquestes nanopartícules específiques són una plataforma prometedora com a nanomedicines pel transport de fàrmacs o sondes pel diagnòstic i tractament d’afliccions relacionades amb CD44. Durant la producció recombinant microbiana, moltes proteïnes heteròlogues tendeixen a acumular-se en cossos d’inclusió. Tenint en compte la última descripció d’aquestes entitats, constitueixen una font de proteïna pràcticament pura. Tot i que s’han desenvolupat tècniques per l’obtenció de proteïna activa mitjançant l’ús de detergents solubilitzadors no desnaturalitzants de manera satisfactòria, és desconegut com aquests procediments afecten el procés d’oligomerització de nanopartícules proteiques. En aquesta tesi, hem analitzat quin impacte té, a nivell estructural i funcional, l’origen de la font proteica. Depenent de si les nanopartícules deriven de la fracció soluble o de la resolubilització dels cossos d’inclusió, s’obtenen diferents organitzacions supramoleculars amb comportaments funcionals diferenciats. Aquests resultats demostren la rellevància que té l’origen cel·lular del material quan parlem d’estructures proteiques complexes. Tot i que recentment s’ha demostrat que la bactèria hoste té una influencia directe en la estructura i el rendiment de les nanopartícules proteiques, es desconeix com els hi afecta l’ús de bactèries amb un perfil genètic diferent com soques deficients en xaperones o lliures de lipopolisacàrids. En aquesta tesi, s’ha investigat l’estructura de nanopartícules dirigides a CD44 o CXCR4 produïdes en diferents soques d’E.coli i com les característiques fisicoquímiques afecten la biointeracció d’aquestes nanopartícules. Els resultats demostres la robustesa dels patrons d’oligomerització d’aquestes nanopartícules dirigides a tumor i la influencia que té el perfil genètic microbià en la proporció de les diferents poblacions oligomèriques en el producte cru. Aquesta variabilitat, intrínseca de la bioproducció, tot i ser imprevisible o incontrolable, constitueix un ventall de nanomaterial. De manera exclusiva es pot seleccionar aquella població de nanopartícules amb les característiques funcionals i fisicoquímiques òptimes considerant l’aplicació per la qual s’han dissenyat.
Recombinant multidomain protein building-blocks have demonstrated to be an appealing biotechnological approach for the development of cell-targeted, nanoscale drug carriers on account of protein functional and architectonic versatility. The colorectal tumor targeting efficacy as well as non-toxicity of T22-empowered nanoparticles validates the potential of the de novo engineering approach developed in our research group. An approach based on end-terminal cationic peptides as pleiotropic tags in self-assembling protein building blocks. In an attempt to explore the flexibility of this approach, we have developed two protein-only nanoparticles that specifically bind CD44-receptor, namely A5G27-GFP-H6 and FNI/II/V-GFP-H6. These designed building-blocks promote the formation of ring-shaped structures which are approximately 14 nm in size, stable in plasma that can internalise cells through endocytosis in absence of cell toxicity. These targeted protein nanoparticles, together with the stability and biodistribution of other tested multifunctional self-assembling protein nanoparticles, are promising platforms for the transport of drugs or imaging agents as nanomedicines for breast cancer or other CD44-linked afflictions. Referring to microbial cell factories, many of the recombinant proteins are likely accumulated in IBs upon induction stress; however, considering non-classical structure description of these protein build-ups they represent an abundant and pure protein source. Although active protein has been obtained from IBs through mild solubilisation procedures, it is still unknown how the recovery of active protein from IBs affects the self-assembling process into protein nanoparticles. Thus, we have analyse the impact on nanoparticle structure and functionality considering the protein material source (nanoparticles from the soluble cell fraction or resolubilised from IBs) of the developed CD44-targeted protein vehicles. We have identified altered supramolecular organisation and consequently distinct in vitro and in vivo performance of cell-targeted protein nanoparticles depending on material’s origin. It is a particularly important aspect regarding recombinant production of smart and complex protein structures Albeit it has been recently known that the bacterial host directly influence the architecture and the performance of the produced protein nanoparticles, how the genetic background of E. coli strains deficient of chaperons or LPS-free affect these protein assemblies has not been addressed. In this thesis we have investigated the fine structure of CXCR4 and CD44 targeted nanoparticles produced in different E. coli strains and how their physicochemical characteristics affect the nanoparticles’ biointeractions. Results illustrate the robustness of self-assembling patterns among tumor-targeted GFP nanoparticles and also the influence of genetic background of the producing cells on shifting the distribution of oligomeric population in the pooled material. The intrinsic variability derived from the bioproduction, although being unpredictable or uncontrollable, offers a scope of nanoparticulated material from where optimal population can be selected considering their ultimate application

A papilomatose bovina é uma doença infectocontagiosa de ocorrência mundial, que assola o rebanho brasileiro, sem qualquer atitude efetiva de controle, e que tem como enfermidades associadas a tumores de bexiga hematúria enzoótica e tumores de trato digestório superior caraguatá, responsáveis por sensíveis perdas para a pecuária. Várias tentativas vacinais têm sido empreendidas com finalidades profiláticas ou terapêuticas, porém sem resultados eficazes. Esta situação se deve a aspectos relacionados à estrutura viral que dificultam uma manipulação eficiente para produção de produtos vacinais. Para que tais dados possam ser obtidos, faz-se necessário uma melhor compreensão da ação das proteínas recombinantes. A clonagem em vetores bacterianos para a expressão e purificação dessas proteínas serve a diferentes propósitos. Entre eles, a produção de insumos imunológicos, como testes diagnósticos ou mesmo vacinas. O presente projeto visou à expressão e purificação da proteína de capsídeo recombinante L2 de BPV-2. A proteína foi expressa em bactéria e tentou-se a purificação por coluna de afinidade; entretanto, problemas na purificação não possibilitaram a conclusão deste objetivo. Todas as abordagens e protocolos utilizados nesse trabalho são discutidos para contribuição ao conhecimento do processo.
The bovine papillomatosis is an infectious disease of worldwide occurrence, plaguing the Brazilian herd, without any effective attitude control, and whose illnesses associated with bladder tumors \"enzootic hematuria\" and upper digestive tract tumors \"caraguatá\" sensitive responsible for losses to livestock. Several attempts have been undertaken vaccine with prophylactic or therapeutic purposes, but without effective results. This is due to issues related to viral structure that hinder efficient manipulation for production of vaccine products. In order to obtain such information, it is necessary better understanding of the action of recombinant proteins. The bacterial cloning vectors for the expression and purification of such proteins serve different purposes. Among them, the production of immune inputs, such as diagnostic tests or vaccines. This project aimed the expression and purification of recombinant L2 capsid protein of BPV-2. The protein was expressed in bacteria and purification was carried out by affinity column. However, difficulties in the purification process, impaired the full completion of this objective. All the attempted approaches and protocols were discussed and potential solutions proposed.

Pyrosequencing is a DNA sequencing method based on thedetection of released pyrophosphate (PPi) during DNA synthesis.In a cascade of enzymatic reactions, visible light isgenerated, which is proportional to the number of nucleotidesincorporated into the DNA template. When dNTP(s) areincorporated into the DNA template, inorganic PPi is released.The released PPi is converted to ATP by ATP sulfurylase, whichprovides the energy to luciferase to oxidize luciferin andgenerate light. The excess of dNTP(s) and the ATP produced areremoved by the nucleotide degrading enzyme apyrase.

The commercially available enzymes, isolated from nativesources, show batch-tobatch variations in activity and quality,which decrease the efficiency of the Pyrosequencing reaction.Therefore, the aim of the research presented in this thesis wasto develop methods to recombinantly produce the enzymes used inthe Pyrosequencing method. Production of the nucleotidedegrading enzyme apyrase by Pichia pastoris expression system,both in small-scale and in an optimized large-scale bioreactor,is described. ATP sulfurylase, the second enzyme in thePyrosequencing reaction, was produced inEscherichia coli. The protein was purified and utilizedin the Pyrosequencing method. Problems associated with enzymecontamination (NDP kinase) and batch-to-batch variations wereeliminated by the use of the recombinant ATP sulfurylase.

As a first step towards sequencing on chip-format,SSB-(single-strand DNA binding protein)-luciferase and KlenowDNA polymerase-luciferase fusion proteins were generated inorder to immobilize the luciferase onto the DNA template.

The application field for the Pyrosequencing technology wasexpanded by introduction of a new method for clone checking anda new method for template preparation prior the Pyrosequencingreaction.

Keywords:apyrase, Pyrosequencing technology, Z_basictag fusion, luciferase, ATP sulfurylase, dsDNAsequencing, clone checking, Klenow-luciferase, SSB-luciferase,Pichia pastoris, Echerichia coli.

The ligand activated transcription factor Aryl Hydrocarbon Receptor (AHR) forms a DNA binding heterodimer with the Aryl Hydrocarbon Nuclear Translocator (ARNT) in response to planar aromatic hydrocarbons. In addition to AHR and ARNT there are at least three other proteins involved in AHR signaling. These proteins are the co-chaperone p23, Ara-9 and two molecules of Heat Shock Protein-90 (HSP-90). This study documents the production of Ara-9 and C∆418 (an ARNT deletion construct) in a modified thioredoxin fusion system. These proteins were expressed in a system that allowed for removal from the fusion partner via a thrombin recognition site as well as the incorporation of an in vitro phosphorylation site. The proteins were then expressed and column purified from E. coli. Once the proteins were expressed and purified they were cleaved from the thioredoxin fusion partner and radioloabeled. Following optimization of the proteolytic digest and radio-labeling each protein was subject to two methods of functional analysis. C∆418 function was assessed by electrophoretic mobility shift assay (EMSA) and proved to effectively form a DNA binding heterodimer with ARNT. In addition the functionality of C∆418 was assessed by co-precipitation showing that the ThioHis-produced C∆418 was indeed able to dimerize with C∆553 (an AHR deletion construct). The ThioHisproduced Ara-9 was also assessed for functionality by EMSA and showed that it was able to restore AHR/ ARNT/DRE complex formation as effectively as Ara-9 produced in a baculovirus system. In addition the function of ThioHis-Ara-9 was also assessed through Far-Western blotting for its ability to associate with renatured HSP-90. These studies involving C∆418 and Ara-9 show that these proteins can be efficiently produced in a functional manner utilizing an inexpensive bacterial system In addition this study documents the production of a plasmid (pCMV-Ara-9) for transfection into the HepG2 cell line to monitor the effects of increased cellular Ara-9 on AHR.

L’aparició de farmacoresistència bacteriana als antibiòtics convencionals és una situació d’alarma mundial. Aquest escenari ha obligat a implementar mesures com la millora de les pràctiques d’higiene o l’administració controlada d’antibiòtics per reduir el seu ús en totes les àrees en les que s’utilitzen comunament, inclosa la medicina humana i animal i la indústria animal de producció d’aliments . Totes aquestes mesures estan destinades a disminuir l’aparició i propagació de la resistència als antibiòtics entre els bacteris, però quan es tracta de combatre als bacteris que ja presenten una o múltiples resistències, es necessiten amb urgència alternatives als antibiòtics. En aquest context, s’han proposat diferents estratègies, entre les quals s’inclouen l’ús de citoquines i pèptids antimicrobians (AMPs). Les citoquines són petites proteïnes reguladores intercel·lulars que tenen un paper central en l’inici, manteniment i regulació de la resposta immune innata. Concretament, l’IFN-γ té un paper fonamental en la promoció de la immunitat protectora contra les infeccions. D’altra banda, els AMPs, com GWH1, són generalment petits pèptids catiònics de naturalesa amfipàtica que tenen activitats antibacterianes, antifúngiques i antivirals d’ampli espectre, la capacitat de modular la resposta immune de l’hoste i una possibilitat reduïda d’induir resistència bacteriana. Malgrat el seu potencial com a agents antiinfecciosos, les citoquines i els AMPs estan subjectes a diversos desavantatges que s’han d’abordar abans de la seva possible aplicació biomèdica. En particular, la baixa estabilitat és un inconvenient comú associat a aquest tipus de compostos proteics. En aquest sentit, s’han aplicat diferents estratègies per superar aquesta limitació i millorar l’eficàcia d’aquests fàrmacs després de la seva administració. La majoria d’aquestes estratègies consisteixen en la vehicularización d’aquestes proteïnes o pèptids en estructures superiors, proporcionant un entorn protector i permetent la possibilitat d’alliberar-los en localitzacions concretes. La formació d’aquestes estructures pot ser modulada a través d’un disseny racional sobre el gen de la proteïna recombinant, com, per exemple, la incorporació de certs pèptids en l’estructura de la proteïna per generar components individuals amb capacitat autoensamblant (nanopartícules solubles), o per obtenir proteïnes propenses a l’agregació (cossos d’inclusió -IBs-). Encara que aparentment diferents, tots aquests complexos s’originen a partir d’interaccions proteiques impulsades per factors termodinàmics i cinètics similars que finalment condueixen a la formació de diferents formats de proteïnes; arranjaments estructurals superiors que involucren a través d’interaccions dirigides o no dirigides, la convergència de formes proteiques aïllades. En aquesta tesi s’ha caracteritzat i avaluat la nanoformulación d’aquestes molècules (citocines i AMPs) en diferents formats proteics incloent IBs i nanopartícules solubles autoensamblants amb l’objectiu de desenvolupar alternatives terapèutiques als antibiòtics. A més, s’ha llançat una mica de llum sobre les forces que governen el procés d’agregació i com es pot modular per promoure la formació d’aquests IBs.
La aparición de farmacorresistencia bacteriana a los antibióticos convencionales es una situación de alarma mundial. Este escenario ha obligado a implementar medidas como la mejora de las prácticas de higiene o la administración controlada de antibióticos para reducir su uso en todas las áreas en las que se utilizan comúnmente, incluida la medicina humana y animal y la industria animal de producción de alimentos. Todas estas medidas están destinadas a disminuir la aparición y propagación de la resistencia a los antibióticos entre las bacterias, pero cuando se trata de combatir a las bacterias que ya presentan una o múltiples resistencias, se necesitan con urgencia alternativas a los antibióticos. En este contexto, se han propuesto diferentes estrategias, entre las que se incluyen el uso de citoquinas y péptidos antimicrobianos (AMPs). Las citoquinas son pequeñas proteínas reguladoras intercelulares que desempeñan un papel central en el inicio, mantenimiento y regulación de la respuesta inmune innata. Concretamente, el IFN-γ tiene un papel fundamental en la promoción de la inmunidad protectora contra las infecciones. Por otro lado, los AMPs, como GWH1, son generalmente pequeños péptidos catiónicos de naturaleza anfipática que tienen actividades antibacterianas, antifúngicas y antivirales de amplio espectro, la capacidad de modular la respuesta inmune del huésped y una posibilidad reducida de inducir resistencia bacteriana. A pesar de su potencial como agentes antiinfecciosos, las citoquinas y los AMPs están sujetos a varias desventajas que deben abordarse antes de su posible aplicación biomédica. En particular, la baja estabilidad es un inconveniente común asociado a este tipo de compuestos proteicos. En este sentido, se han aplicado diferentes estrategias para superar esta limitación y mejorar la eficacia de estos fármacos tras su administración. La mayoría de estas estrategias consisten en la vehicularización de estas proteínas o péptidos en estructuras superiores, proporcionando un entorno protector y permitiendo la posibilidad de liberarlos en localizaciones concretas. La formación de estas estructuras puede ser modulada a través de un diseño racional sobre el gen de la proteína recombinante, como, por ejemplo, la incorporación de ciertos péptidos en la estructura de la proteína para generar componentes individuales con capacidad autoensamblante (nanopartículas solubles), o para obtener proteínas propensas a la agregación (cuerpos de inclusión -IBs-). Aunque aparentemente diferentes, todos estos complejos se originan a partir de interacciones proteicas impulsadas por factores termodinámicos y cinéticos similares que finalmente conducen a la formación de diferentes formatos de proteínas; arreglos estructurales superiores que involucran a través de interacciones dirigidas o no dirigidas, la convergencia de formas proteicas aisladas. En esta tesis se ha caracterizado y evaluado la nanoformulación de estas moléculas (citoquinas y AMPs) en diferentes formatos proteicos incluyendo IBs y nanopartículas solubles autoensamblantes con el objetivo de desarrollar alternativas terapéuticas a los antibióticos. Además, se ha arrojado algo de luz sobre las fuerzas que gobiernan el proceso de agregación y cómo se puede modular para promover la formación de dichos IBs.
The emergence of bacterial drug resistance to conventional antibiotics is a global alarming situation. This worrying scenario has forced the implementation of measures such us improved hygiene practices or antibiotic stewardship to reduce antimicrobial usage in all areas in which these therapeutics are commonly used, including human and animal medicine and food-producing animal industry. All these measures are intended to diminish the appearance and spread of drug resistance among bacteria, but when it comes to combat against drug or multidrug resistant bacteria, alternatives to traditional antibiotics are urgently needed. In this context, different strategies have been proposed as promising alternatives to antibiotics, including the use of cytokines and antimicrobial peptides (AMPs). Cytokines are small intercellular regulatory proteins that play a central role in initiating, maintaining, and regulating the innate immune response. Specifically, IFN-γ has a pivotal role in promoting protective immunity against infections. On the other hand, AMPs such as GWH1, are generally small cationic peptides with an amphipathic nature that have a broad-spectrum antibacterial, antifungal and antiviral activities, the ability to modulate the host immune response and a reduced possibility of inducing bacterial drug resistance. Despite their potential as anti-infective agents, cytokines and AMPs are subjected to several disadvantages that must be addressed prior to their possible biomedical application. In particular, low stability is a common drawback associated to these type of protein compounds. In this sense, different technologies and strategies have been applied in order to overpass this limitation and improve the efficiency of these drugs after administration. Most of these strategies consist on the vehicularization of these proteins or peptides into superior complexes, providing a protective environment and allowing the possibility of deliver them to the target site. The formation of these superior structures may be modulated by a direct rational design over the recombinant protein gene, such as que incorporation of certain peptides into the protein structure to generate building blocks for spontaneous self-assembling (soluble nanoparticles), or to obtain prone-to-aggregate proteins (inclusion bodies -IBs-). Although seemingly different, these small-scale complexes all originate from fundamental protein interactions and are driven by similar thermodynamic and kinetic factors finally leading to the formation of different proteins formats; superior structural arrangements that involve through directed or not directed interactions, the convergence of isolated protein forms. In this thesis, the nanoformulation of these molecules (cytokines and AMPs) into different protein formats including IBs and soluble self-assembling nanoparticles have been characterized and evaluated with the aim to develop therapeutic alternatives to antibiotics. In addition, some light has been shed on the forces that govern the aggregation process and how it can be modulated to promote the formation of IBs.
Universitat Autònoma de Barcelona. Programa de Doctorat en Biotecnologia

O P. vivax é a segunda espécie mais prevalente causadora de malária no mundo. Medidas de controle ineficientes exigem o desenvolvimento de novas estratégias de prevenção, como vacinas, novas drogas e novos inseticidas. O objetivo geral do trabalho foi gerar uma formulação vacinal universal com proteínas e adenovírus recombinantes capazes de induzir anticorpos contra as diferentes formas alélicas da proteína circumsporozoíta (CSP) do P. vivax. As proteínas foram produzidas em E. coli e purificadas por cromatografia de afinidade e troca iônica. A obtenção destas proteínas nos permitiu testar qual seria a melhor formulação vacinal para a indução de anticorpos contra as três formas alélicas da proteína CSP de P. vivax (PvCSP). Anticorpos específicos reconheceram esporozoítas do P. vivax por imunofluorescência. Por fim testamos o uso de dois adenovírus recombinantes, um símio e um humano, deficientes em replicação, expressando as três regiões imunodominantes da proteína PvCSP em fusão. Estes foram capazes de induzir resposta imune específica contra as proteínas PvCSP sendo testados em esquema de prime-boost heterólogo, onde camundongos foram primados com os adenovírus e nas doses-reforço receberam a mistura com as três proteínas recombinantes.
The Plasmodium vivax is the second most prevalent species of malaria in the world. Inefficient measures of control used today demand the development of new strategies for prevention, as vaccines, new drugs and new insecticides. The central objective of this thesis was to generate a universal vaccine formulation with proteins and recombinant adenoviral vectors representing the different allelic forms of the circumsporozoite protein (CSP) of the P. vivax. The recombinant proteins were expressed in E. coli and purified. These proteins allowed us to test which would be the best vaccine formulation for the induction of antibodies against the three allelic forms of CSP. The specific antibodies also recognized P. vivax sporozoites by immunofluorescence. Finally we test the use of two recombinant adenoviral vectors, a simian and a human, both replication deficient, expressing a protein containing the repeat regions of the CSP in fusion. These adenoviral vectors induced specific immune response against CSP and were successfully used in an immunization regimen of heterologous prime and boost where in the first dose the mice received recombinant adenoviral vector and in the subsequent doses, the mixture with three recombinant proteins.

L’ús cada cop més extensiu de plataformes “òmiques” per a l’estudi de llevats ha generat grans quantitats d’informació relativa a la fisiologia cel·lular sota diferents condicions de cultiu, la qual pot ser utilitzada per al desenvolupament de noves estratègies d’enginyeria genètica. S’ha observat que els nivells d’expressió d’un fragment d’anticòs humà produït en el llevat metilotròfic Pichia pastoris augmenten significativament quan aquest és cultivat en nivells de baixa disponibilitat d’oxigen. Anàlisis transcriptòmics mostren que hi ha una regulació important dels gens involucrats en el metabolisme de lípids i la resposta al mal plegament de proteïnes (UPR), destacant la l’augment de transcrits provinents de gens involucrats en les rutes de biosíntesi de l’ergosterol i esfingolípids. A més, s’ha observat que la presència en el medi de cultiu de fluconazol, un agent antifúngic que inhibeix específicament l’activitat de la esterol C14α-demetilasa (Erg11p) en la ruta de biosíntesi de l’ergosterol, resulta en una disminució dels nivells d’ergosterol en les membranes cel·lulars, millorant els nivells de proteïna recombinant secretada al medi de cultiu. Finalment, el factor de transcripció Hac1, amb un paper important en la regulació de la UPR, també es troba involucrat en la regulació de la síntesi de lípids. En el present estudi, la soca de referència, P. pastoris que produeix el fragment d’anticòs 2F5 de manera recombinant, s’ha modificat genèticament per tal de canviar la seva composició lipídica. S’ha generat una bateria de soques que presenten alguns gens de la ruta de biosíntesi d’esfingolípids delecionats (DES1, SUR2), o bé sobreexpressats (DES1). Al mateix temps, s’ha alterat la ruta de síntesi de l’ergosterol de la soca de referència mitjançant la sobreexpressió del gen ERG11 o tractant les cèl·lules amb fluconazol. Finalment, també es va sobreexpressar HAC1. Les cèl·lules es van cultivar en cultius en continu sota condicions d’oxigen normals (normoxia), i baixos (hipòxia), i les soques van ser caracteritzades en quant a nivells de Fab produïts, la seva distribució intra/extracel·lular, morfologia a partir d’estudis de microscòpia electrònica (TEM), i el seu contingut lipídic, quantificant els nivells d’àcids grassos, fosfolípids, esterols, lípids neutres i esfingolípids. Amb tota aquesta informació es pretén comprendre com les cèl·lules s’adapten a canvis genètics i ambientals a través de canvis en el seu contingut lipídic. Els resultats obtinguts s’han combinat amb informació transcriptòmica prèviament publicada, demostrant que existeix una important remodelació del metabolisme lipídic quan les cèl·lules es troben en condicions d’hipòxia.
The increasing availability of omics databases provides an important knowledge base for the design of novel yeast engineering strategies, since they offer systems-level information on the physiology of the cells under diverse growth conditions and genetic backgrounds. In a previous study, a beneficial effect of low oxygen availability on the expression of a human Fab fragment in Pichia pastoris has been identified. Transcriptomic analyses pointed out important regulation of the Unfolded Protein Response (UPR) and lipid metabolism, with a significant transcriptional up-regulation of ergosterol and sphingolipid biosynthetic pathways. Furthermore, cells cultured in the presence of fluconazole, an antifungal agent that inhibits sterol C14α-demethylase (Erg11p) activity in the ergosterol biosynthetic pathway, showed a reduced amount of ergosterol in the plasma membrane, resulting in increased protein secretion levels. Moreover, it is known that the transcription factor Hac1, which plays a key role by regulating the UPR, regulates lipid biosynthesis In the present study, a reference strain of P. pastoris secreting the 2F5 Fab antibody fragment as a model recombinant protein was genetically modified in order to change its lipid composition. A set of strains harbouring either deleted (DES1, SUR2) or overexpressed (DES1) genes of the sphingolipid pathway were generated. In addition, a Fab producing strain overexpressing HAC1 or ERG11 were constructed and, as an alternative to the ergosterol pathway knockout, the reference strain was treated with fluconazole. The series of strains were cultivated in chemostat cultures under normoxic and hypoxic conditions. Strains were characterised in terms of Fab- productivity, intra/extracellular product distribution, cell morphology by transmission electron microscopy (TEM) and lipid content in terms of phospholipids, fatty acids, sterols, non-polar lipids and sphingolipids, in order to fully understand how cells adapt to genetic and environmental changes involving lipid changes. The obtained results were combined with previously published transcriptional data, allowing us to demonstrate an important remodelling of the lipid metabolism under limited oxygen availability.

True biospecific affinity chromatography, immobilized metal affinity chelating (IMAC) and ion exchange chromatography (IEX) strategies, were investigated for EBA purification of a model system, recombinant glutathione-S-transferase (GST). All three strategies were performed using an unclarified E.coli homogenate containing GST concentration at 20-25 % of the total protein, which represented a large initial degree of purity. True biospecific affinity strategy exploits the naturally occurring affinity between GST and its natural substrate, glutathione (GSH). EBA purification using STREAMLINE GSH adsorbent was very specific and lead to a high purification fold of the target GST. However, it was hampered by a poor dynamic capacity (39 U mL^-1) and a low recovery yield (34 %). Although some improvements by using a greater volume of adsorbent in the expanded bed and operating at a lower superficial velocity with concomitant lower degrees of bed expansion were obtained, the overall purification performance remained unsatisfactory. This supports the hypothesis that affinity adsorbents are an unnecessary elaboration where products are already substantially pure. The use of short peptide tags on proteins in conjunction with EBA, using generic adsorbents to facilitate the facile recovery of the target protein, is a promising approach to simplify downstream processing flowsheets. Two IMAC adsorbents, STREAMLINE-IDA and STREAMLINE-NTA loaded with Ni²⁺ were used to purify GST-(His)₆, a construct specifically tagged with a histidine-rich sequence at the C-terminus. Both adsorbents exhibited adsorption performance similar to each other in EBA purification. They remained superior with high dynamic adsorption capacities (IDA at 357 U mL^-1 and NTA at 263 U mL^-1), high selective separation and highly robust adsorbents, compared to IEX and GSH adsorption systems. However, if the target protein is destined for therapeutic use, it may be necessary to incorporate additional processes to ensure the removal of the histidine-rich tag sequence. Due to the propensity of GST to form multimers, cleavage of the tag from this protein was unsuccessful. Such complications may disfavour the widespread adoption of this approach. However, in other model protein, polyhistidine tagged maltose-binding protein (MBP), successful tag digestion was obtained. The tag removal process was simple and efficient with a high yield of MBP. The simplicity and ability to operate with minimal loss of yield, indicates that the combination of affinity fusions and IMAC strategy for EBA, has great potential for efficient large-scale operation. The use of economic I EX approaches is an attractive alternative to avoid the need for affinity tag fusion consideration. IEX are popular for protein purification due to their robustness and versatility. When using STREAMLINE DEAE, additional purification steps are required to achieve a high degree of product purity, due to their low protein selectivity and some competition for adsorption between GST and  Other Components  in  the feedstock.

Recombinant Protein G (rPG), an engineered form of streptococcal protein G with a theoretical molecular weight of 22.26 kDa was successfully cloned and expressed in E.coli BL 21(DE3) cells. The albumin binding domain was removed during the gene synthesis to avoid unspecific binding. This recombinant form of protein G contains only the IgG binding domains along with the 6X histidine tag at the N terminal. The removal of non-specific domains maximizes the specificity of IgG binding through the Fc region. The recombinant protein G was purified through heat treatment and using immobilized metal affinity chromatography (IMAC). On an SDS-PAGE gel there was only a single band of the purified preparation which migrated at 32 KDa, however when analyzed by mass spectrometry it was ~22.4 kDa, this phenomenon of retarded migration on SDS-PAGE has been known from previous studies. The production of recombinant protein has also been optimized. The effects of expression temperature, inducer type, inducer concentration and media composition have been investigated. The expression was done at 10 liter scale using the best expression conditions, and the protein was purified to homogeneity, dialyzed and lyophilized. The pure protein was immobilized on a POROS AL (Self Pack® POROS® 20 AL, Applied Biosystems, USA). The immobilized protein IgG binding has been monitored using a VersAFlo system. This system allowed real time monitoring of IgG binding characteristics.

 

Therapeutic recombinant proteins such as antibodies, hormones, enzymes, and anticoagulants can be used in medicine, for food production, agriculture and bioengineering industries. Recombinant proteins are currently being produced by mammalian, plant, insect and microbial culture systems but microalgae, when used as an expression vector, would offer many benefits over existing methods. These advantages include, lower production costs, faster growth rates, easier culturing, simpler transgenic manipulation as well as modified abilities of transcription and translation. South Africa boasts one of the most bio-diverse regions globally, with tremendous species richness in freshwater eco-systems with the potential of commercially desired, novel microalgae species. To investigate the feasibility of establishing microalgal biotechnology in South Africa, the model green microalgae species, Chlamydomonas reinhardtii was transformed under available conditions. Furthermore, five hundred microalgal isolates previously collected across South Africa and recorded as part of the Microalgae Culture Collection held at the Council for Scientific and Industrial Research (CSIR), were bio-prospected for their growth potential. First a gene encoding the Chlamydomonas codon optimised green fluorescent protein (CrGFP) was cloned into a pChlamy4 vector under the control of the chimeric Hsp70A-rbcS2 promoter and selected for with a bleomycin resistant gene, she ble. The verified plasmid was then electro-transformed into the nuclear region of two C. reinhardtii strains CC-125 and CC-400. The microalgae transformants were assessed at the DNA level by PCR and further at the CrGFP protein production level by fluorescence spectrophotometry. From the Microalgae Culture Collection Database, forty isolates were chosen microscopically based on whether they are unicellular, non-colonial, with large or many chloroplasts and a thin cell wall. The isolates underwent a growth curve analysis and those with the best growth rates were then directly compared to C. reinhardtii spp. under phototrophic and mixotrophic growth conditions. These isolates were then identified by Sanger sequencing of the 18S rRNA region. Results indicated successful insertion of the Crgfp transgene with detectable levels of CrGFP fluorescence above autofluorescence for all transformed C. reinhardtii colonies. In order of the greatest growth potential, microalgae with high growth rates were identified as C. sorokiniana, Chlorella sp., C. vulgaris, T. obliquus, T. dimorphus and R. subcapitata species. In this study, the South African Chlorella spp. isolates had a greater growth rate in both phototrophic and mixotrophic medium than the C. reinhardtii controls. In this study a foundation was laid for microalgae biotechnology research at the CSIR in South Africa, with C. reinhardtii as the reference organism; as well as considering the potential nuclear transformation in select indigenous species, such as Chlorella sp. for future microalgae protein expression systems.
Dissertation (MSc)--University of Pretoria, 2019.
NRF DST Innovation and Priority Research Areas Masters Scholarship
Plant Production and Soil Science
MSc
Unrestricted

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第20450号
農博第2235号
新制||農||1050(附属図書館)
学位論文||H29||N5071(農学部図書室)
京都大学大学院農学研究科森林科学専攻
(主査)教授 杉山 淳司, 教授 髙部 圭司, 教授 梅澤 俊明
学位規則第4条第1項該当

El llevat metilotròfic Pichia pastoris (genere Komagataella) s’ha convertit en una de les plataformes cel·lulars més populars per produir proteïnes d’interès industrial, la majoria de les quals es secreten extracel·lularment per facilitar el posterior procés de purificació. La secreció de proteïnes heteròlogues fora de la cèl·lula està mediada per la via secretora, una ruta que engloba diversos passos i té diferents orgànuls interconnectats per aconseguir una secreció eficient. Tanmateix, quan P. pastoris es veu obligat a produir proteïnes heteròlogues, el correcte funcionament de la via de secreció podria veure’s compromesa. En aquest context, s’han realitzat diferents estudis per identificar colls d’ampolla específics i maximitzar la producció de proteïnes. Es va identificar la translocació de proteïnes del citoplasma al reticle endoplasmàtic (ER) com un coll d’ampolla precoç important que es troba a la via secretora. El pas de translocació sol estar mediat per un pèptid senyal que es troba al N-terminal de la proteïna a traduir. Depenent de la seqüència senyal, aquest pas es pot realitzar mentre la proteïna es tradueix (translocació co-traduccional) o després de la traducció (post-traducció). A més, la senyal de secreció ha de contenir l’anomenada regió ‘pro’ per mediar una ràpida exportació de la proteïna recombinant des del ER al Golgi per tal que s’acabi secretant. A la primera part d’aquest estudi, ens vam centrar en l’enginyeria i la caracterització del pèptid senyal de secreció com a estratègia per millorar la secreció de proteïnes recombinants. Per tal de comparar-les, hem utilitzat el senyal de secreció de Saccharomyces cerevisiae anomenat ‘alfa mating factor’ (α-MF) que és el senyal de secreció més comú utilitzat per la comunitat científica per secretar proteïnes recombinants en P. pastoris. Es diu que aquest senyal de secreció condueix proteïnes mitjançant la traducció post-traduccional. Com a resultat, si el α-MF es fusiona amb una proteïna que es pot plegar en el citosol del llevat, és possible que la proteïna no pugui travessar el translocó del ER i entrar a la via secretora. La solució va ser substituir la seqüència senyal pre-α MF per la seqüència senyal pre-Ost1, que dirigeix la translocació de forma co-traduccional a través del ER, garantint així que les proteïnes heteròlogues només es pleguin després d’arribar al lumen del ER. Addicionalment, la pro-regió del α-MF contenia una regió propensa a l’agregació, que va ser fàcilment eliminada mitjançant l’intercanvi d’una treonina per una serina en la posició 42 (Ser42). Aquest senyal de secreció híbrid resultant va augmentar dràsticament la producció de tres proteïnes models diferents utilitzades en aquesta tesi: una proteïna model fluorescent anomenada E2-Crimson i dues lipases diferents d’interès industrial anomenades Lipasa 2 de Bacillus thermocatenulatus (BTL2) i Lipasa de Rhizopus oryzae (ROL). Més important encara, es van provar aquestes troballes a escala bioreactor, obtenint així resultats similars als observats a escala matràs. En particular, les soques amb el senyal de secreció millorat van tenir un millor rendiment cel·lular en comparació amb l’α-MF. En resum, s’ha proposat un nou senyal de secreció híbrid per substituir el senyal de secreció α MF com a estàndard predeterminat per produir proteïnes heteròlogues en P. pastoris. No obstant això, per aprofitar plenament el potencial del senyal de secreció millorat, es necessita enginyeria cel·lular addicional per superar els colls d’ampolla que apareixen posteriorment de la translocació, particularment en condicions de bioprocessos com en un fed-batch.
La levadura metilotrófica Pichia pastoris (genero Komagataella) se ha convertido en una de las plataformas celulares más populares para producir proteínas de interés industrial, la mayoría de las que se secretan extracelularmente para facilitar el posterior proceso de purificación. La secreción de proteínas heterólogas fuera de la célula está mediada por la vía secretora, una ruta que engloba varios pasos y tiene diferentes orgánulos interconectados para conseguir una secreción eficiente. Sin embargo, cuando P. pastoris se ve obligada a producir proteínas heterólogas, el correcto funcionamiento de la vía de secreción podría verse comprometida. En este contexto, se han realizado diferentes estudios para identificar cuellos de botella específicos y maximizar la producción de proteínas. Se identificó la translocación de proteínas del citoplasma al retículo endoplasmático (ER) como un cuello de botella precoz importante que se encuentra en la vía secretora. El paso de translocación suele estar mediado por un péptido señal que se encuentra en el N-terminal de la proteína a traducir. Dependiendo de la secuencia señal, este paso se puede realizar mientras la proteína se traduce (translocación co-traduccional) o después de la traducción (post-traduccional). Además, la señal de secreción debe contener la llamada región “pro” para mediar una rápida exportación de la proteína recombinante desde el ER al Golgi para que acabe secretando. En la primera parte de este estudio, nos centramos en la ingeniería y la caracterización del péptido señal de secreción como estrategia para mejorar la secreción de proteínas recombinantes. Con el fin de compararlas, hemos utilizado la señal de secreción de Saccharomyces cerevisiae llamado “alfa mating factor” (α-MF) que es la señal de secreción más común utilizado por la comunidad científica para secretar proteínas recombinantes en P. pastoris. Se dice que esta señal de secreción conduce proteínas mediante la traducción post-traduccional. Como resultado, si el -MF se fusiona con una proteína que se puede plegar en el citosol de la levadura, es posible que la proteína no pueda atravesar el translocón del ER y entrar en la vía secretora. La solución fue sustituir la secuencia señal pre-α MF por la secuencia señal pre-Ost1, que dirige la translocación de forma co-traduccional a través del ER, garantizando así que las proteínas heterólogas sólo se plieguen después de llegar al lumen del ER. Adicionalmente, la pro-región del α-MF contenía una región propensa a la agregación, que fue fácilmente eliminada mediante el intercambio de una treonina por una serina en la posición 42 (Ser42). Esta señal de secreción híbrido resultante aumentó drásticamente la producción de tres proteínas modelos diferentes utilizadas en esta tesis: una proteína modelo fluorescente llamada E2-Crimson y dos lipasas diferentes de interés industrial llamadas Lipasa 2 de Bacillus thermocatenulatus (BTL2) y lipasa de Rhizopus oryzae (ROL). Más importante aún, se probaron estos hallazgos a escala biorreactor, obteniendo así resultados similares a los observados a nivel matraz. En particular, las cepas con la señal de secreción mejorado tuvieron un mejor rendimiento celular en comparación con la α-MF. En resumen, se ha propuesto una nueva señal de secreción híbrido para sustituir la señal de secreción α-MF como estándar predeterminado para producir proteínas heterólogas en P. pastoris. Sin embargo, para aprovechar plenamente el potencial de la señal de secreción mejorado, se necesita ingeniería celular adicional para superar los cuellos de botella que aparecen posteriormente de la translocación, particularmente en condiciones de bioprocesos como en un fed-batch.
The methylotrophic yeast Pichia pastoris (Komagataella spp.) has become one of the most popular cellular platforms to produce industrially relevant proteins, most of which are secreted in the extracellular media to facilitate subsequent downstream processing. The secretion of heterologous proteins out of the cell is mediated by the secretory pathway, a route that encompasses several steps and has different organelles interconnected to achieve an efficient secretion. However, when P. pastoris is forced to produce heterologous proteins, the proper function of the secretion pathway might be compromised. In this context, different studies have been carried out to identify specific bottlenecks and maximize protein production. We identified the translocation of proteins from the cytoplasm to the Endoplasmic Reticulum (ER) as a major early bottleneck present in the secretory pathway. The translocation step is usually mediated by a signal sequence present at the N-terminal of the protein to be translocated. Depending on the signal sequence, this step can be performed while the protein is being translated (co-translational translocation) or after the translation (post-translational translocation). In addition, the secretion signal has to contain a so-called pro-region to mediate a fast export of the recombinant protein from the ER to the Golgi for subsequent secretion. In the first part of this study, we focused on the engineering and characterization of the secretion signal peptide as a strategy to improve recombinant protein secretion. For comparison, we used the secretion signal from the alpha mating factor (α-MF) from Saccharomyces cerevisiae, which is the most common secretion signal used by the scientific community to secrete recombinant proteins in P. pastoris. This secretion signal is reported to drive proteins through the post-translational translocation. As a result, if the α-MF is fused to a protein that can fold in the yeast cytosol, the protein may be unable to traverse the ER translocon and enter the secretory pathway. The solution was to replace the pre-α MF signal sequence with the pre-Ost1 signal sequence, which directs co-translational translocation across the ER membrane, thereby ensuring that heterologous proteins fold only after reaching the ER lumen. Additionally, the pro-region from the α-MF contained a region prone to aggregation, which was easily removed by exchanging a threonine by a serine at position 42 (Ser42). The resulting hybrid secretion signal drastically increased the production of three different model proteins used in this thesis: a fluorescent model protein called E2-Crimson and two different lipases of industrial interest, namely the lipase 2 from Bacillus thermocatenulatus (BTL2) and a lipase from Rhizopus oryzae (ROL). More importantly, these findings were then tested at bioreactor scale, thereby obtaining similar results to those observed at shake flask scale. Notably, strains with the improved secretion signal had a better cell performance in comparison with the α-MF. Overall, a new hybrid secretion signal has been proposed to replace the α factor secretion signal as the default standard for producing heterologous proteins in P. pastoris. However, to fully exploit the power of the improved secretion signal, additional cellular engineering is needed to overcome bottlenecks that appear downstream of the translocation event, particularly under bioprocess (fed-batch) conditions.

Non-pathogenic lactic acid bacteria are economically important Gram-positive bacteria used extensively in the food industry. Due to their “generally regarded as safe” status, certain species from the genera Lactobacillus and Lactococcus are also considered desirable as candidates for the production and secretion of recombinant proteins, particular those with therapeutic applications. The hypothesis examined by this thesis is that Lactococcus lactis can be modified to be an effective antimicrobial agent. Therefore, the aims of this thesis were to investigate the optimisation of the expression, secretion and/or activities of potential heterologous antimicrobial proteins by the model lactic acid bacterium, Lactococcus lactis subsp. cremoris MG1363. L. lactis strains were engineered to express and secrete the recombinant CyuC surface protein from Lactobacillus reuteri BR11, and a fusion protein consisting of CyuC and lysostaphin using the Sep promoter and secretion signal. CyuC has been characterised as a cystine-binding protein, but has also been demonstrated to have fibronectin binding activity. Lysostaphin is a bacteriolytic enzyme with specific activity against the Gram-positive pathogen, Staphylococcus aureus. These modified L. lactis strains were then investigated to see if they had the ability to inhibit the adhesion of S. aureus to host extracellular matrix (ECM) proteins. It was observed that the cell extracts of the L. lactis strain with the vector only (pGhost9:ISS1) was able to inhibit the adhesion of S. aureus to fibronectin, whilst the cell extracts of the L. lactis strain expressing lysostaphin was able to inhibit adhesion to keratin. Finally, this thesis has identified specific lactococcal genes that affect the secretion of lysostaphin through the use of random transposon mutagenesis. Ten mutants with higher lysostaphin activity contained insertions in four different genes encoding: (i) an uncharacterised putative transmembrane protein (llmg_0609), (ii) an enzyme catalysing the first step in peptidoglycan biosynthesis (murA2), (iii) a homolog of the oxidative defence regulator (trmA), and (iv) an uncharacterised putative enzyme involved in ubiquinone biosynthesis (llmg_2148). The higher lysostaphin activity observed in these mutants was found to be due to higher amounts of lysostaphin being secreted. The findings of this thesis contribute to the development of this organism as an antimicrobial agent and also to our understanding of L. lactis genetics.

Metabolic flux analysis (MFA) is a powerful technique for quantifying the intracellular fluxes in central carbon metabolism. It relies on detection of stable isotope labelling from metabolites such as amino acids derived from protein. Current standard techniques are, however, unable to distinguish between different cell types in heterogeneous tissue. The aim of the thesis was to address this problem by developing and validating a strategy using green fluorescent protein (GFP) with cell type specific expression as a reporter protein for investigating the fluxes in specific cell types in the Arabidopsis thaliana root. The fundamental difficulty in applying a reporter protein strategy in a multicellular organism arises from the limited amount of recombinant protein expressed by the cells. The main novel contributions of the work in this thesis are threefold. First, a robust protocol for purification of GFP from the roots of Arabidopsis seedlings and for detection of reliable mass isotopomer distributions from the amino acids derived from GFP are described. Secondly, the reporter protein strategy is validated in this biological system with a focus on showing the data obtained by the use of the reporter protein is equal to that normally obtained from the total protein fraction. To expand on this, stable isotope labelling in isolated root hair cells is explored. These cells are easily isolated and show potential as a model system for cell type specific metabolism. Finally, the experimental data provide evidence for the feasibility of measuring data from specific cell types with appropriate mass spectrometric techniques. Analysis of cell type specific gene expression in this system suggests differences in the primary metabolism of different cell types cannot be ruled out without further investigation. Based on small scale in silico modelling described in this thesis, new solutions capable of providing data on sub-populations of cells are required, if central metabolism of the cell types differs significantly.

Brucella abortus is capable of resisting the microbicidal mechanisms of phagocytic cells and growing within phagocytic cells, usually macrophages. B. abortus, like several other intracellular bacteria responds to the hostile environment in macrophages by producing heat shock proteins (HSPs) which are induced by environmental stresses. Bacterial HSPs are very immunogenic, eliciting both cellular and humoral immune responses in the infected host. The significance of host cellular and protective immune responses directed against these proteins is currently unresolved. Baculovirus recombinants were generated in Sf9 insect cells for B. abortus HSPs and the protein expression was optimized. Humoral (Western blot), cell mediated (CMI, IFN-g- release by splenocytes, and CD3+CD4+, CD3+CD8+ T cell/ total splenocytes ratios) and protective immune responses of BALB/c mice (challenge with virulent B. abortus 2308) against these recombinants, against B. abortus superoxide dismutase (SOD) and ribosomal L7/L12 proteins, inoculated alone or in various combinations with complete Freund's, Ribi and recombinant IL-12 as adjuvants, were analyzed. Vaccinia virus-GroEL recombinant as priming immunogen, followed by baculovirus-GroEL-Ribi booster, was explored. Androstenediol, an immune up-regulator, was tested for its ability to induce resistance against challenge. None of the mice inoculated with individual, divalent or trivalent HSP-expressing Sf9 cells combined with Freund's were protected against challenge and the Sf9 cell-induced response masked the recombinant protein-specific CMI responses. Recombinant HSPs were purified and combined with Ribi. Although significant IFN-g release was induced by immunization with the HtrA-Ribi combination, no mice were protected against challenge. Priming with vaccinia virus-GroEl recombinant and boosting with purified baculovirus-GroEL protein-Ribi combination did not induce protection. Androstenediol did not enhance in vivo resistance to challenge. IL-12 alone did not activate splenocytes but induced significant IFN-g release in mice when combined with killed B. abortus RB51 vaccine, purified recombinant HtrA or purified SOD proteins, or L7/L12 expressing Escherichia coli cells. Significant protection was induced by SOD combined with IL-12. No correlation was seen between IFN-g release by splenocytes and protection against challenge in the SOD/IL-12-immunized mice. The results suggest that B. abortus HSPs are not highly immunogenic in mice and though various immune responses may be induced by one or another HSPs, protective immune response, unfortunately, is not among them. The results of this study reflect the difficulties in experimenting with immune responses against single or a limited number of recombinant B. abortus proteins. This is particularly true when the task includes induction of a protective immune response and finding significant correlation between different types of immune response assays.
Ph. D.

La present tesi doctoral es centra en la caracterització de sistemes d’expressió utilitzats per a la producció de proteïnes recombinants (RPP) en el llevat metilotròfic Pichia pastoris. Al llarg de tot aquest treball s’integren els resultats de diferents camps -enginyeria de bioprocessos i regulació génica- per tal d’ampliar la informació sobre els sistemes d’expressió analitzats i, així, reduir la incertesa a l’dissenyar un bioprocés RPP. En el primer capítol de la tesi, s’estudia el sistema clàssic d’expressió basat en el PAOX1. En un primer pas, es van cultivar en quimiòstat dos clons productors de la Lipasa 1 de Candida rugosa (Cr1l) amb diferent dosi gènica. D’aquesta manera, es va determinar la interrelació de tres factors importants en els bioprocessos RPP com ara la velocitat específica de creixement (μ), els nivells d’expressió relativa de el gen heteròleg (RTL) i la velocitat específica de generació de producte (qp) . A més, també es va monitoritzar l’expressió d’un factor de transcripció important per a la via d’utilització de metanol (MIT1). Un cop establertes les condicions òptimes d’operació en continu, es van dur a terme cultius en fed-batch per validar i comparar el comportament dels clons productors observat, tant des del punt de vista del seu estat fisiològic com de la cinètica de producció de Crl1 . Els inconvenients de l’ús de el potent sistema d’expressió basat en PAOX1-principalment derivat de l’ús de l’metanol com a font de carboni, font d’electrons i inductor de RPP- han portat a la comunitat de Pichia a investigar i desenvolupar sistemes d’expressió alternatius que no depenguin de l’addició de metanol. A causa d’això, en el segon capítol de la tesi, es van caracteritzar dos nous sistemes d’expressió, basats en el PPDF i PUPP, per determinar si poden competir amb l’àmpliament utilitzat PGAP en la producció de la Lipasa B de Candida antarctica (Calb) . El comportament dels tres sistemes d’expressió es va comparar, en primer lloc, en quimiòstat. Això va permetre obtenir informació sobre la influència de la μ i l’expressió de Calb en la cinètica de producció de la proteïna. A més, els clons productors de la proteïna recombinant sota els nous sistemes d’expressió es van cultivar a fed-batch, en les condicions òptimes observades prèviament, per tal de provar la potencial escalabilitat del bioprocés. En l’últim capítol de la tesi es va optimitzar, utilitzant cèl·lules senceres de P. pastoris (whole cells), la producció del citrocromo P450 2C9 humà (CYP2C9). La coexpressió d’aquest enzim d’interès industrial al costat del domini donador d’electrons (cytochrome P450 reductase, CPR) es va realitzar mitjançant un nou sistema de promotors bidireccionals. Després d’una fase de screening en la qual es van provar fins a 8 promotors per a la producció de CYP2C9 / CPR, es va seleccionar la combinació que va proporcionar la major activitat oxidasa, i es van realitzar una sèrie de cultius per optimitzar el bioprocés. D’aquesta manera, es va determinar la influència de paràmetres importants de l’bioprocés -μ, pH i estratègia d’addició de metanol en la producció de biocatalizador. Finalment, l’eficiència de l’biocatalizador obtingut es va testar en la reacció de hidroxilació de l’ibuprofèn.
La presente tesis doctoral se centra en la caracterización de sistemas de expresión utilizados para la producción de proteínas recombinantes (RPP) en la levadura metilotrófica Pichia pastoris. A lo largo de todo este trabajo se integran los resultados de diferentes campos —ingeniería de bioprocesos y regulación génica— con el fin de ampliar la información sobre los sistemas de expresión analizados y, así, reducir la incertidumbre al diseñar un bioproceso RPP. En el primer capítulo de la tesis, se estudia el sistema clásico de expresión basado en el PAOX1. En un primer paso, se cultivaron en quimiostato dos clones productores de la Lipasa 1 de Candida rugosa (Cr1l) con diferente dosis génica. De esta manera, se determinó la interrelación de tres factores importantes en los bioprocesos de RPP tales como la velocidad específica de crecimiento (µ), los niveles de expresión relativa del gen heterólogo (RTL) y la velocidad específica de generación de producto (qp). Además, también se monitorizó la expresión de un factor de transcripción importante para la vía de utilización del metanol (MIT1). Una vez establecidas las condiciones óptimas de operación en continuo, se llevaron a cabo cultivos en fed-batch para validar y comparar el comportamiento de los clones productores observado, tanto desde el punto de vista de su estado fisiológico como de la cinética de producción de Crl1. Los inconvenientes del uso del potente sistema de expresión basado en PAOX1—principalmente derivado del uso del metanol como fuente de carbono, fuente de electrones e inductor de RPP— han llevado a la comunidad de Pichia a investigar y desarrollar sistemas de expresión alternativos que no dependan de la adición de metanol. Debido a ello, en el segundo capítulo de la tesis, se caracterizaron dos nuevos sistemas de expresión, basados en el PPDF y PUPP, para determinar si pueden competir con el ampliamente utilizado PGAP en la producción de la Lipasa B de Candida antarctica (CalB). El comportamiento de los tres sistemas de expresión se comparó, en primer lugar, en quimiostato. Esto permitió obtener información sobre la influencia de la µ y la expresión de CALB en la cinética de producción de la proteína. Además, los clones productores de la proteína recombinante bajo los nuevos sistemas de expresión se cultivaron en fed-batch, en las condiciones óptimas observadas previamente, con el fin de probar la potencial escalabilidad del bioproceso. En el último capítulo de la tesis se optimizó, utilizando células enteras de P. pastoris (whole cells), la producción del citrocromo P450 2C9 humano (CYP2C9). La coexpresión de esta enzima de interés industrial junto al dominio donador de electrones (cytochrome P450 reductase, CPR) se realizó mediante un novedoso sistema de promotores bidireccionales. Después de una fase de screening en la que se probaron hasta 8 promotores para la producción de CYP2C9/CPR, se seleccionó la combinación que proporcionó la mayor actividad oxidasa, y se realizaron una serie de cultivos para optimizar el bioproceso. De este modo, se determinó la influencia de parámetros importantes del bioproceso —µ, pH y estrategia de adición de metanol— en la producción de biocatalizador. Finalmente, la eficiencia del biocatalizador obtenido se testó en la reacción de hidroxilación del ibuprofeno.
The present thesis is focused on the characterization of alternative expression systems used for recombinant protein production (RPP) in the methylotrophic yeast Pichia pastoris. Over this whole work, the integration of results from different fields —bioprocess engineering and gene regulation— is attempted in order to fill the gaps that frequently come up when designing a RPP bioprocess. In the first chapter of the thesis, the classical PAOX1-based expression system is thoroughly studied. Specifically, through a set of chemostat cultivations of two clones expressing the Candida rugosa lipase 1 (Cr1l) with different gene dosage, it was determined the interrelation of three important factors in RPP processes such as specific growth rate (µ), heterologous gene relative transcript levels (RTL) and specific product generation rate (qp). Moreover, the expression of a crucial transcription factor of the methanol utilization pathway (MIT1) was also determined in chemostat cultivations. Once the optimal operation conditions were identified in steady state conditions, fed-batch cultivations were conducted to validate the clones’ behaviour observed previously in terms of both physiological state and Crl1 production kinetics. The inherent drawbacks of using the powerful PAOX1-based expression system —mainly derived from the use of methanol as carbon source, electron source and RPP inducer— has forced the Pichia community to investigate and develop alternative methanol-free expression systems. Due to that, in the second chapter of the thesis, two novel expression systems, based on the PPDF and PUPP, were similarly characterized in order to determine whether they can compete in terms of protein production with the widely used PGAP, usually considered the methanol-free reference, for the production of the Lipase B from Candida antarctica (CalB). All the three expression systems performance was firstly compared in chemostat cultivations, which enabled to shed light on the influence of µ and CALB expression on the CalB production kinetics. Additionally, the clones harboring the novel expression system were cultivated in fed-batch mode at the optimal conditions observed in chemostat in order to test their potential bioprocess scalability. Finally, in the last chapter of the thesis, the production of active whole cell biocatalyst based on the human cytochrome P450 2C9 (CYP2C9) in P. pastoris was afforded. The coexpression of the protein along with its redox partner (cytochrome P450 reductase, CPR) was achieved by means of a bidirectional promoter system. After a screening phase in which up to 8 promoters were tested for CYP2C9/CPR production, the combination that provided the best balance was selected for subsequent bioprocess optimization experiments. In this way, the influence of important bioprocess parameters — pH, µ and methanol addition— on active CYP2C9/CPR whole cell biocatalyst production was determined. Finally, the efficiency of P. pastoris whole cell biocatalyst based on CYP2C9/CPR was tested in a proof of concept reaction of interest, in which ibuprofen is hydroxylated into its oxidized derivatives.
Universitat Autònoma de Barcelona. Programa de Doctorat en Biotecnologia

Many biopharmaceutical projects require the production of recombinant protein in a bacterial host. Conventional procedures used for recombinant protein production (RPP) involve the rapid synthesis of the target protein. This results in the accumulation of unfolded protein, the induction of the heat shock stress response and bacterial growth arrest. More importantly, the target protein accumulates in inclusion bodies and hence is useless to determine its structure. The immediate impact of this is that both the yield and quality of the target protein are compromised. This thesis reports two generically successful approaches that were developed to overcome this series of stress-induced events in Escherichia coli. Both strategies were developed during the production of a cytoplasmic protein and outer membrane lipoproteins using the pET expression system in the bacterial host, E. coli strain BL21(DE3)*. First, the induction protocol was modified to minimise the stress on the host bacterium. This method relies on the induction of very low levels of the T7 RNA polymerase in BL21* and thus the correspondingly slow synthesis of the target protein. Using this approach, growth and productivity of different types of correctly folded target proteins were sustained for at least 70 h. Secondly, mutant hosts that significantly improve recombinant protein production during conventional protocols were isolated. These improved hosts are resistant to IPTG-induced stress and continue to accumulate high levels of the correctly folded target protein. Key to the stress resistance is the presence of mutations that downregulate the synthesis of T7 RNA polymerase. However, different improved hosts were able to enhance the production of different types of target protein, such as those requiring extensive post-translational modification. The potential for isolating a plethora of improved bacterial hosts that are tailored for the production of different types of recombinant protein is discussed in light of the challenges faced by bioindustry. Procedures enabling the isolation of mutant hosts during the production of GFP-tagged and untagged proteins are reported.

Microalgae are considered as attractive platforms for the synthesis of high-value heterologous proteins due to their many beneficial attributes including ease of cultivation, lack of pathogenic agents, and low-cost downstream processing. However, recombinant protein levels are low compared to microbial platforms and commercial production is not a reality yet. Promising research using the model microalga Chlamydomonas reinhardtii has highlighted this potential, particularly for transgene expression in the chloroplast. The objective of this research was to study different strategies for expression in the C. reinhardtii chloroplast aimed at increasing growth rate and recombinant protein production, by means of cell engineering and bioprocess optimisation. Two main approaches were considered for this purpose. Firstly, the insertion of the light-harvesting protein proteorhodopsin (PR) into the cell membrane, that has been reported to increase growth rate and culture lifespan in bacterial systems. This has resulted in PR accumulation to low yet detectable levels providing a moderate increase (12 %) of specific cell growth. Noteworthy, this is the first demonstration of an integral membrane recombinant protein expressed in the chloroplast. The second approach was to express a gene for a novel fluorescent protein (VFP) under the control of different regulatory elements. Detectable VFP levels were produced, with increased protein levels when using the psaA promoter/5’UTR element, and with co-expression of a gene for the Spy chaperone. These strains were used to study the effect of temperature, media and light intensity on recombinant protein production and cell growth. Protein levels and fluorescence allowed determining improved cultivation conditions as 30 °C under mixotrophic mode, and these conditions were tested for the accumulation of a therapeutic protein (Cpl-1 endolysin). In conclusion, protein productivity was observed to be protein-specific and improved conditions that increase protein levels for one protein cannot necessarily be extrapolated to the accumulation of a different protein.

Tobacco has been studied as a host for producing recombinant therapeutic proteins on a large-scale, commercial basis. However, the proteins expressed in tobacco usually need to be purified to high yield and purity from large amounts of biomass in order for their production to be commercially viable. The methods needed to purify proteins from tobacco are very challenging and not well studied. The objective of this research was to develop a process for the purification of the acidic model protein, recombinant β-glucuronidase (rGUS), from transgenic tobacco leaf tissue to high yield and purity.

Polyelectrolyte precipitation with polyethyleneimine (PEI) was identified as an initial purification step for purifying acidic recombinant proteins from tobacco. Polyethyleneimine precipitation allowed for high recovery and concentration of the target protein while removing large amounts of impurities from the initial extract. At dosages of 700-800 mg PEI/g total protein, nearly 100% of the rGUS activity was precipitated with generally more than 90% recovered from the pellet. In addition, more than 60% of the native tobacco proteins were removed in the process, resulting in a purification factor near 4.

Recombinant GUS was further purified by a step of hydrophobic interaction chromatography (HIC) followed by a step of hydroxyapatite chromatography (HAC). The HIC step served to remove PEI and other contaminants such as nucleic acids that were accumulated during the precipitation step, while the HAC step served to separate rGUS from the remaining native tobacco proteins, most notably ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco). Nearly 40% of the initial rGUS activity was recovered as a near homogeneous fraction based on SDS-PAGE analysis after the three step process.

The main steps used in this process are anticipated to be scalable and do not rely on affinity separations, making the process potentially applicable to a wide variety of acidic recombinant proteins expressed in tobacco as well as other leafy crops.
Master of Science

Relaxin is a small peptide hormone that has demonstrated potential therapeutic actions for cardiovascular disease and fibrosis. Additionally, relaxin has demonstrated the ability to protect the heart from injuries caused by ischemia and reperfusion, promote the healing of ischemic ulcers, and counteract allergic responses. The objective of this research was to express fully processed porcine relaxin in transgenic tobacco plants, as an alternative to current methods of producing relaxin.

Two recombinant relaxin genes were constructed that contained the patatin signal peptide cDNA fused in frame to prorelaxin cDNA, which was codon-optimized for expression in Nicotiana tabacum, under the control of either the â superâ promoter or the dual enhanced cauliflower mosaic virus 35S promoter. Eighteen transgenic tobacco plants were generated that were transformed with the above recombinant genes. Preprorelaxin, mRNA was detected in 12 of the transgenic plants. Fully processed relaxin protein was not found in any tobacco plants that had demonstrated gene expression by northern blot analysis. Preprorelaxin was only identified in extracts from transgenic plants that contained the insoluble protein fraction, as determined by western blot analysis. Additionally, an increased yield of preprorelaxin was identified after incubation of tobacco leaves in an ubiquitin inhibitor.
Master of Science

Bibliography: leaves 182-198. Improvements in the current production system of inclusion bodies and the downstream processing sequence are essential to maintain a competitive advantage in the market place. Optimisation of fermentation is considered to improve production yield; then flotation as a possible inclusion body recovery method.

Biomaterials requirements nowadays are becoming more and more specialized to meet increasingly demanding needs for biomedical applications such as matrices for tissue scaffolds. Among various useful classes of biomaterials, protein-based materials have been extensively pursued as they can offer a wide range of material properties to accommodate a broader spectrum of functional and performance requirements. The advent of genetic engineering and recombinant DNA technology has enabled the production of new protein-based biopolymers with precisely controlled amino acid sequence. As an example, silk-elastinlike protein (SELP) polymers consisting of polypeptide sequences from native silk of remarkable mechanical strength and polypeptide sequences from native elastin that is extremely durable and resilient have been produced. In this dissertation, a particular silk-elastinlike protein copolymer, SELP-47K, was cast into film form, and fully characterized for its material properties, including the mechanical property, secondary structure transition, optical transparency, surface, and other physical, chemical properties. The relationship between mechanical property and protein secondary structure was investigated as well. In addition, the material property tunability which can be induced by physical, mechanical, and chemical treatments has been explored. It is worth noting that the physically crosslinked SELP-47K films displayed mechanical properties comparable to those of native elastin obtained from bovine ligament. Secondary structure study through Raman and FTIR spectra showed that methanol treatment is capable of inducing theβ-sheet crystallization of silklike blocks, which act as physical crosslinks in the protein polymer chain network, thus stabilizing the protein structure and conferring the improved material integrity. The SELP-47K protein polymer thin films displayed excellent optical transparency. In particular, its excellent optical transmittance (over 90%) in visible light range may indicate SELPs can be a family of promising biomaterial candidate for ocular applications. Besides material property characterization, SELP-47K protein polymer has been fabricated into a variety of drug delivery devices to sustainably release a common ocular antibiotic, ciprofloxacin over a period of up to 220 h, with near-first order kinetics.

The optimisation of recombinant protein production by animal cell cultures is important for the economic feasibility of these processes. Simultaneously with product yield, product authenticity is a crucial aspect to consider as it may per se affect the therapeutic value of such proteins. More defined culture media are being developed, particularly to ensure batch product consistency. A Chinese Hamster Ovary cell line (CHO 320) producing human interferon-γ (IFN-γ), a glycosylated protein, was chosen to investigate the effects of the culture environment on (I) cell growth, (2) product yield and (3) product authenticity. A statistical approach was used to identify important culture components for cell growth and IFN-γ production. When the concentration of the resulting positive variables was initially increased in culture, improvements of approximately 40% in both of these parameters were achieved; the glycosylation of IFN-γ was not affected. The former analysis also indicated that different stimuli were required for growth and production. Fed-batch feeding of glucose and glutamine, components depleted early from culture, did not prolong cell growth or IFN-γ production but the initial glycosylation pattern of IFN-γ was a function of glutamine concentration. Bovine serum albumin (BSA) was shown to have important role(s) in culture and cell growth was not possible in its absence. Pluronic F68, alone or in combination with a lipid mixture or linoleic acid, was able to restore cell growth in low BSA (1 mg/ml) cultures. However, IFN-γ production was significantly reduced and the extent of IFN-γ glycosylation also changed. These effects were related to: (1) BSA concentration, (2) BSA type, and ultimately, (3) lipid composition of the culture. The results reported in this thesis exhibit the necessity to consider the effects of the culture environment not only on cell growth and product yield but also on product authenticity throughout any optimisation process.

Pichia pastoris s’ha convertit en una de les plataformes cel·lulars més utilitzades per a la producció de proteïnes recombinants i metabòlits d’alt valor afegit. En els darrers anys s’han aconseguit fites importants en l’anàlisi quantitativa a nivell de sistemes de la seva fisiologia. Aquesta gran quantitat d’informació ha permès desenvolupar models metabòlics a escala genòmica, que permeten el desenvolupament de noves estratègies per l’enginyeria de soques i de bioprocés. Amb anterioritat a aquest estudi s’havien publicat tres models metabòlics a escala genòmica per a P. pastoris. No obstant això, aquests models presentaven algunes inconsistències en algunes vies metabòliques, en la nomenclatura de metabòlits i reaccions, així com les anotacions associades a certes vies. A més, algunes de les rutes metabòliques o característiques específiques de P. pastoris eren representades de forma errònia o incompleta. És per això que en aquest estudi es desenvolupa un model metabòlic a escala genòmica consens, que integra els models anteriors. A més, també es fa una revisió exhaustiva de diverses rutes metabòliques i nombroses vies es corregeixen i actualitzen d’acord amb les publicacions disponibles. Com a resultat, el nou model, iMT1026, pot reproduir amb més precisió els paràmetres de creixement experimentals de cèl·lules creixent en glucosa i diferents nivells de disponibilitat d’oxigen. Amb la voluntat d’expandir les capacitats del model, es generen noves dades fisiològiques fent servir dos dels substrats més importants per aquesta factoria cel·lular. Es realitzen unes series de cultius en continu per a la caracterització del perfil fisiològic de P. pastoris creixent en glicerol i metanol com a fonts úniques de substrat. A més, també es caracteritza la composició macromolecular de la biomassa. Posteriorment, s’incorporen en el model noves equacions de biomassa específiques per a cada font de carboni. Aquestes noves dades experimentals han permès estimar els paràmetres energètics associats a les fonts de carboni i validar el model (iMT1026 v3.0) per aquestes condicions de creixement. Tot i la validació de iMT1026 v3.0 en un rang més ampli de condicions, en aquest treball es prova en dues aplicacions diferents: en l’anàlisi de fluxos metabòlics basat en 13C i com a eina de suport per a la interpretació de resultats en soques amb modificades en el metabolisme redox. Tot i que hi ha un únic estudi on s’analitza la relació entre fluxos metabòlics en cèl·lules creixent en glicerol, no es té constància de cap estudi d’anàlisi de fluxos metabòlics en aquesta font de carboni. Així doncs, es redueix el model metabòlic a escala genòmica a un model del metabolisme central i es fa servir per a l’anàlisi de fluxos metabòlics basats en 13C en cèl·lules creixent amb glicerol a diferents velocitats de creixement. Els resultats obtinguts són molt consistents amb els cultius previs en glicerol. També s’utilitza iMT1026v3.0 com a suport per a la interpretació del perfil fisiològic obtingut en soques amb el metabolisme redox modificat. Una soca que expressa un fragment d’anticòs es modifica genèticament mitjançant l’expressió d’una NADH quinasa, de manera que el balanç de cofactors redox queda pertorbat. Les soques generades mostren una producció de proteïna recombinant més elevada i una alteració en el perfil macroscòpic de creixement. Mitjançant l’anàlisi in silico dels perfils fisiològics resultants, es prediuen possibles canvis metabòlics associats a l’alteració del balanç de cofactors que estan d’acord amb el perfil macroscòpic observat. Així doncs, en línies generals, en aquest treball es desenvolupa una eina precisa per a l’enginyeria de sistemes metabòlics. A més, és validada en condicions vàries i s’utilitza en dues aplicacions diferents que demostren la seva fiabilitat.
Pichia pastoris has become one of the most extensively used platform cell factories for recombinant protein and high-value added metabolite production. In the past recent years, important breakthroughs in the systems-level quantitative analysis of its physiology have been achieved. This wealth of information has allowed the development of genome-scale metabolic models, which make new approaches possible for host cell and bioprocess engineering. Previous to this work, three different genome-scale metabolic models were available for P. pastoris. Nevertheless, these models showed some inconsistencies regarding certain pathways, including the terminology for both metabolites and reactions and annotations. Furthermore, some P. pastoris specific metabolic traits were misrepresented. Therefore, in this study, a consensus genome-scale metabolic model has been developed, thereby integrating the prior models. In addition, a comprehensive revision of metabolic pathways was performed and several pathways were curated and updated according to the currently available literature. As a result, the new model, iMT1026, is able to more accurately reproduce experimental growth parameters using glucose as carbon source and different oxygen availability conditions. In order to expand the capabilities of the consensus model, new physiological datasets of cells growing on two of the most relevant substrates for this cell factory were generated. Specifically, a series of chemostat cultivations were performed to characterise the physiologic profile and macromolecular biomass composition of P. pastoris growing on glycerol and methanol as sole carbon sources. Also, macromolecular biomass composition was analysed, allowing us to incorporate new carbon-source specific stoichiometric biomass equations into the model, as well as to estimate the associated energetic parameters. Overall, a new version of the model (iMT1026 v3.0) was validated for these growing conditions. In addition to the validation of iMT1026 v3.0 for a wider range of carbon sources and growth conditions, we have further tested its performance in two different applications, namely, the generation of reduced metabolic models suitable for 13C-based metabolic flux analysis and, assisting the interpretation of physiological growth parameters of redox-cofactor engineered strains. In particular, the genome-scale metabolic model has been reduced into a core model and used for 13C-based metabolic flux analysis of cells growing on glycerol at different growth rates. To our knowledge, this is the first study ever reported of 13C-MFA using glycerol as sole carbon source. Notably, flux analyses are highly consistent with pioneering 13C-based metabolic profiling studies of P. pastoris growing on glycerol. iMT1026 v3.0 was also employed for assisting the interpretation of the physiological profiles obtained for redox-cofactor engineered strains. A recombinant strain producing an antibody fragment was engineered to overexpress a heterologous NADH kinase, aiming at increased NADPH regeneration rates. Notably, the redox-engineered strains showed an increase in recombinant protein production and altered macroscopic growing profiles. In silico analysis of the impact of NADH kinase overexpression using the iMT1026 model predicted possible metabolic changes associated to the redox cofactor imbalance that were in agreement with the observed physiological phenotypes. Overall, a refined tool for systems metabolic engineering is provided in the present study. Moreover, such tool has been validated for a wide range of environmental conditions and employed in two different applications, confirming its reliability.

La plataforma desenvolupada d’enginyeria de proteïnes auto-acoblables permet dissenyar nanopartícules únicament proteiques (NPs) capaces d’atacar i actuar selectivament sobre les cèl.lules canceroses mitjançant la interacció amb receptors que es sobreexpressen. Les estructures esfèriques estables de les NPs desenvolupades i la seva mida adequada, en combinació amb els pèptids d’orientació , milloren la seva especificitat. A més, la incorporació de segments de toxina i verí ha millorat els efectes terapèutics d’aquestes estructures que són totalment biocompatibles i que no tenen cap portador extern o material agregat, complint d’aquesta manera amb el nou concepte per a medicaments de precisió, que involucra un fàrmac recombinant lliure de vehicle, auto-acoblat, auto-dirigit i eficient. Una versió modificada de la cadena catalítica ricina A, amb la capacitat de disminuir els efectes secundaris no desitjats de la síndrome de vessament vascular però conservant la seva citotoxicitat natural, es va adaptar a la plataforma de proteïnes. El disseny es va desenvolupar amb el pèptid T22, que s’uneix a CXCR4, en l’extrem N-terminal, i una cua d’histidines a la terminal-C, en combinació amb un fragment del lloc escindible de furina per alliberar la proteïna intracel.lularment, i una seqüència KDEL per evitar la secreció del reticle endoplàsmic. Les NPs de cadena de ricina A solubles purificades dirigides a CXCR4 amb un diàmetre mitjà de 11 nm, van assolir un increment de 100 vegades en la seva citotoxicitat amb un IC50 de 13 ± 0,5 x 10 -9 M en cèl.lules HeLa. També es van produir per mètodes recombinants i es van purificar cossos d’inclusió insolubles de 400-600 nm, amb resultats citotòxics parcials. El mecanisme d’entrada dependent del receptor d’T22-MRTA-H6 es va verificar i avaluar en un model de ratolí amb leucèmia mieloide aguda (AML) mitjançant la injecció sistèmica a la vena de la cua on es va verificar un bloqueig important de les cèl.lules leucèmiques sense toxicitat sistèmica o histològica lateral en els òrgans sans. De manera similar, la clorotoxina (CTX) també es va incorporar a la plataforma de proteïnes per tal d’aprofitar la seva d’orientació i efecte terapèutic en glioblastoma (GBM), ambdues funcions en un sol pèptid. Es van dissenyar dues versions que s’uneixen a la proteïna anexina-2 i la metaloproteinasa de matriu MMP-2; CTX-GFP-H6 i CTX-KRKRK-GFP-H6. Les NPs solubles d’un diàmetre mitjà de 12 nm es van incubar en cèl·lules HeLa, sobreexpressant annexina-2, i en cèl.lules U87MG, sobreexpressant MMP2. Les dues versions eren completament fluorescents, però CTX-GFP-H6 va presentar efectes citotòxics lleus, mentre que CTX-KRKRK-GFP-H6 va mostrar ser més citotòxic en les cèl.lules U87MG que en les cèl.lules HeLa. L’afinitat selectiva de CTX es va confirmar mitjançant l’avaluació de la seva selectivitat utilitzant anticossos monoclonals i un sèrum policlonal contra la proteïna de la superfície cel.lular, actuant com un receptor de la CTX.
La plataforma desarrollada de ingeniería de proteínas autoensamblables permite diseñar nanopartículas únicamente proteicas (NPs) capaces de atacar y actuar selectivamente sobre las células cancerosas mediante la interacción con receptores que se sobreexpresan. Las estructuras esféricas estables de las NPs desarrolladas y su tamaño adecuado, en combinación con los péptidos de direccionamiento involucrados, mejoran su especificidad. Además, la novedosa incorporación de segmentos de toxina y veneno ha mejorado los efectos terapéuticos de estas estructuras que son totalmente biocompatibles y que no tienen ningún portador externo o material agregado, cumpliendo de esta manera con el concepto emergente para medicamentos de precisión que involucra un fármaco recombinante libre de vehículo, autoensamblado, auto-dirigido y eficiente. Una versión modificada de la cadena catalítica de ricina A, con la capacidad de disminuir los efectos secundarios no deseados del síndrome de derrame vascular, pero conservando su citotoxicidad natural, se adaptó a la plataforma de proteínas. El diseño se desarrolló con el péptido T22, que se une a CXCR4, en el extremo N-terminal, y una cola de histidinas en el extremo C-terminal, en combinación con un fragmento del sitio escindible de furina para liberar la proteína intracelularmente, y una secuencia KDEL para evitar secreción del retículo endoplásmico. Las NPs de cadena de ricina A solubles purificadas dirigidas a CXCR4, con un diámetro promedio de 11 nm, alcanzaron un incremento de 100 veces en su citotoxicidad con un IC50 de 13 ± 0,5 x 10 -9 M en células HeLa. Pero también se produjeron por métodos recombinantes y se purificaron cuerpos de inclusión insolubles de 400-600 nm, con resultados citotóxicos parciales. El mecanismo de entrada dependiente del receptor de T22-mRTA-H6 se verificó y evaluó en un modelo de ratón con leucemia mieloide aguda (AML) mediante la inyección sistémica en la vena de la cola, donde se verificó un bloqueo importante de las células leucémicas sin toxicidad sistémica o histológica lateral en los órganos sanos. De manera similar, la clorotoxina (CTX) también se incorporó a la plataforma de proteínas con el fin de aprovechar su direccionamiento y efecto terapéutico en glioblastoma (GBM), ambas funciones en un solo péptido. Se diseñaron dos versiones que se unen a la proteína anexina-2 y la metaloproteinasa de matriz MMP-2; CTX-GFP-H6 y CTX-KRKRK-GFP-H6. Lss NPs solubles, de un diámetro promedio de 12 nm, se incubaron en células HeLa sobreexpresando anexina-2, y en células U87MG, sobreexpresando MMP2. Ambas versiones eran completamente fluorescentes, pero CTX-GFP-H6 presentó efectos citotóxicos leves, mientras que CTX-KRKRK-GFP-H6 mostró ser más citotóxico en las células U87MG que en las células HeLa. La afinidad selectiva de CTX se confirmó mediante la evaluación de su direccionamiento utilizando anticuerpos monoclonales y un suero policlonal contra la proteína de la superficie celular, actuando como un receptor de la CTX.
The developed self-assembling platform allows the engineering of protein-only nanoparticles (NPs) capable to target and act selectively over cancer cells by means of the interaction with overexpressed receptors. The stability of the spherical NP structures and their adequate size, in combination with the involved targeting peptides, enhance their specificity. Also, the novel incorporation of toxin and venom segments have improved the therapeutic effects of these fully biocompatible materials, without the need of any external carrier or added material, thus fulfilling the newfangled concept for precision medicines that involve self-assembled, self-targeted and efficient vehicle-free recombinant drugs. A modified version of the catalytic ricin A chain, with the ability to diminish the undesired vascular leak syndrome side effects but retaining its natural cytotoxicity, was adapted to the protein platform. The design was developed with the peptide T22 in the N-terminal, which binds CXCR4, and a his-tag in the C-terminal. This was combined with a furin cleavable site fragment in order to release the protein intracellularly, and a KDEL sequence to avoid endoplasmic reticulum secretion. Purified soluble CXCR4-targeted ricin A chain NPs with an average diameter of 11 nm, reached a 100-fold cytotoxic improvement with an IC50 of 13 ± 0.5 x 10 -9 M in HeLa cells. Also, insoluble 400-600 nm inclusion bodies were produced by recombinant methods and purified, with partial cytotoxic results. The receptor-dependent mechanism of T22-mRTA-H6 was verified and evaluated in an acute myeloid leukemia (AML) mouse model by systemic administration through a vein tail injection where an important blockage of the leukemic cells was verified without side systemic or histological toxicity in healthy organs. In a similar way, chlorotoxin (CTX) was also incorporated to the protein platform in order to take advantage of its targeting and therapeutic effect in glioblastoma (GBM), both functions in one peptide. Two versions that target protein Annexin-2 and the matrix metalloproteinase MMP-2 were engineered, namely CTX-GFP-H6 and CTX-KRKRK-GFP-H6. The soluble NPs of an average dimeter of 12 nm were incubated with HeLa cells, overexpressing annexin-2, and in U87MG cells, overexpressing MMP2. Both versions were fully fluorescent but CTX-GFP-H6 presented mild cytotoxic effects, whereas CTX-KRKRK-GFP-H6 showed to be more cytotoxic in U87MG cells than in HeLa cells. The selective affinity of CTX was confirmed by means of evaluating its targeting using a monoclonal antibody and a polyclonal serum against the cell surface protein, acting as a CTX receptor.

El llevat metilotròfic Pichia pastoris (Komagataella sp.) és un dels sistemes d’expressió més atractius per a la producció de proteïna recombinant, mercat contínuament en expansió. El fort promotor del gen de l’alcohol oxidasa 1 (PAOX1), induït per metanol però reprimit per glucosa, glicerol o etanol, és un dels més emprats per aquest propòsit. No obstant, existeixen encara diversos colls d’ampolla fisiològics que limitant el procés. En aquest context, diferents estratègies han estat proposades i provades per tal de millorar la producció heteròloga de molts tipus diferents de proteïna. Les aproximacions més habituals inclouen increment en el nombre de còpies de gen heteròleg, enginyeria de promotors i modificació dels mecanismes de plegament i secreció. L’objectiu d’aquesta tesi ha estat el desenvolupament de noves estratègies per incrementar la producció de proteïna recombinant, emprant la lipasa de Rhizopus oryzae (Rol) com a proteïna model en el sistema d’expressió basat en el PAOX1. Primerament, els gens del factors de transcripció de PAOX1, MXR1 i MIT1, es van sobreexpressar constitutivament per tal de millorar la transcripció de ROL. Això es va confirmar degut a una millora en la capacitat assimilatòria de metanol i un increment en els nivells relatius de mRNA de ROL i varis gens relacionats amb el metabolisme del metanol, i.e. revertint l’efecte de titulació causat per la transcripció de múltiples cassettes d’expressió de ROL. Tot i aquestes millores, els nivells extracel·lulars d’activitat lipàsica no van augmentar de forma significativa en cultius en quimiòstat, apuntant a colls d’ampolla addicionals limitant la producció de Rol. En segon lloc, es van explorar possibles dianes d’enginyeria metabòlica en el metabolisme cel·lular de P. pastoris emprant el model metabòlic a escola genoma (GEM) consens iMT1026 v3.0. Aquest pas in silico va proporcionar diversos knock-outs prometedors que serien experimentalment testats fent servir el sistema d’edició genòmica CRISPR/Cas9. Les simulacions apuntaven a la disponibilitat de NADPH i una limitada aportació de determinats aminoàcids (serina i cisteïna) com a potencials factors limitants de la producció de Rol. Una reducció en el fitnes cel·lular que afecta a la viabilitat de les soques que es buscaven obtenir va impedir la verificació de la majoria dels knock-outs proposats. Finalment, donat que les nostres anàlisis i estudis prèviament publicats identificaven el NADPH com un cofactor important limitant la producció de proteïna recombinant, els nostres esforços es van dirigir a incrementar la seva disponibilitat a través d’estratègies de knock-in de gens. Específicament, vam sobreexpressar dos gens que codificaven per enzims redox, una NADH quinasa i una NADH oxidasa, amb l’objectiu de pertorbar directament l’equilibri redox de la cèl·lula. A més, es va comprovar l’efecte fisiològic d’aquests enzims fent servir diferents mescles co-substrat/metanol com a font de carboni. En resum, vam observar un increment en la producció de proteïna recombinant amb diferents graus de millora depenent de la font de carboni provada. També vam realitzar anàlisis transcriptòmiques i una avaluació in silico dels nostres resultats per tal de presentar una interpretació millor de l’estat fisiològic de la cèl·lula. Dins del nostre coneixement, aquest és el primer estudi dirigit a incrementar la generació de NADPH en un sistema d’expressió basat en PAOX1, en condicions de creixement en metanol. A grans trets, noves estratègies d’enginyeria de soques han estat proposades i provades durant l’execució d’aquest estudi. A més a més, s’han aplicat GEMs i aproximacions relacionades amb biologia de sistemes, demostrant que són eines potents i prometedores per al disseny racional d’organismes industrials.
The methylotrophic yeast Pichia pastoris (Komagataella sp.) is one of the most attractive expression systems for heterologous protein production, which constitutes a continuously expanding market. The strong alcohol oxidase gene 1 promoter (PAOX1), induced by methanol but repressed by glucose, glycerol or ethanol, is one of the most used for this purpose. Nevertheless, there still exist several physiological bottlenecks limiting the process. In this context, several strategies have been proposed and tested in order to improve the heterologous production of many different types of proteins. Common approaches include increasing heterologous gene copy number, promoter engineering and modification of the folding and secretory mechanisms. The aim of this thesis has been the development of new strategies to increase recombinant protein yields, using the Rhizopus oryzae lipase (Rol) as model protein in a PAOX1-based expression system. Firstly, the PAOX1 transcription factor genes MXR1 and MIT1 were constitutively overexpressed aiming at improving ROL transcription. This was confirmed by an improved methanol assimilation capacity and an increase in relative mRNA levels of ROL and several genes related with methanol metabolism, i.e. reverting the titration effect caused by the transcription of multiple ROL expression cassettes. Despite such improvements, extracellular lipase activity levels did not increase significantly in chemostat cultures, pointing out to additional bottlenecks limiting Rol production. Second, possible metabolic engineering targets in P. pastoris’ cell metabolism were explored using the consensus genome-scale metabolic model (GEM) iMT1026 v3.0. This in silico step provided several promising knock-outs which were going to be experimentally tested using the CRISPR/Cas9 genome editing system. The simulations pointed to NADPH availability and limited supply of some amino acids (serine and cysteine) as potential Rol production limiting factors. A reduction in cell fitness affecting the viability of the obtained strains impeded to verify most of the proposed knock-outs. Finally, since our in silico analyses and previously published studies identified NADPH as an important limiting cofactor in recombinant protein production, our efforts were geared towards increasing its availability through gene knock-in strategies. Specifically, we overexpressed two genes encoding redox enzymes, a NADH kinase and a NADH oxidase, with the aim to directly perturb the cell’s redox balance. Further, we tested the physiological effect of these enzymes using different co-substrate/methanol mixtures as carbon source. In short, we observed an increase in recombinant protein production with different degrees of improvement depending on the carbon source(s) tested. We also performed a transcriptomic analysis and an in silico evaluation of our results in order to provide a better interpretation of the cell physiological state. To our knowledge, this is the first study aiming to increase NADPH generation in the PAOX1-based expression system, under methanol growth conditions. Overall, novel strain engineering strategies have been proposed and tested during the execution of this study. Furthermore, GEMs and related systems biology approaches were applied, proving to be promising powerful tools for rational engineering of industrial microorganisms.

When the cell is under stress arising from oxidation, heat, infection, toxic contamination or any other stressful condition, proteins may unfold and expose residues in their structure that under normal physiological conditions are hidden and shielded from chemical reactions.

In this licentiate thesis the effects of general oxidative stress on the production of recombinant protein by mammalian cells are considered.

The work consisted of a broad literary review focused on oxidative stress and cellular response, cross-protection, gene regulation in response to oxidative stress and the relevance of this to pharmaceutical industry. A series of oxidative stressors is described and examined for experimental use. Experimental cultivation and maintenance of several mammalian cell lines was performed and several candidate stressing agents were proven on these cell lines. Menadione was selected as a powerful and consistent stressing agent, and so several experiments were performed where batches of cells were exposed to varying degrees of stress.

The performance of the cells in regard to production of recombinant protein was then examined by ELISA, showing a strong downregulation of production under stressful conditions. Recombinant protein taken from stressed and control cultures is then isolated, purified and examined with MALDI-TOF spectrometry. Finally mRNA from the cells is isolated and examined by means of microarray. Genes that are significantly regulated are examined, and those genes that may have significance in the area of stress regulation and reaction are listed.

The results of the study show that mitomycin C exerts oxidative stress on the industrial protein expressing mammalian cell lines tested.