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1
Bruns, Caroline 1984. "GRh1-dependent unconventional protein secretion." Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/125070.
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A part de la secreció convencional, en la qual les proteïnes són transportades a través del reticle endoplasmàtic (RE) i de l'aparell de Golgi, s'han descrit, per a diferents proteïnes i en organismes diversos, rutes de secreció no-convencional que circumval·len l'aparell de Golgi. No obstant, aquests mecanismes de secreció no estan ben entesos. Se sap que per a la secreció no-convencional de la proteïna d'unió a acil-CoA Acb1 a Saccharomyces cerevisiae són necessàries diverses proteïnes, com ara l'ortòleg de GRASP Grh1, proteïnes relacionades amb l'autofàgia, proteïnes involucra-des en la fusió de membranes amb els endosomes, membres de la maquinària ESCRT, i la t-SNARE de la membrana plasmàtica Sso1. La manera per la qual totes aquestes proteïnes cooperen per a dur a terme la secreció d'Acb1 és encara una incògnita.
Els resultats de les nostres investigacions indiquen que sota inani-ció de nutrients, condició que indueix la secreció no-convencional d'Acb1, Grh1 es concentra en estructures membranoses prop dels llocs de sortida del RE, de manera dependent de fosfatidilinositol-3-cinasa. Aquestes membranes, en forma de tassa, estan enriqui-des en PI(3)P i contenen la proteïna d'ESCRT-I Vps23 així com les proteïnes d'autofàgia Atg8 i Atg9, reunint així diverses proteïnes necessàries per a la secreció d'Acb1. Hem batejat aquestes estructures CUPS (per les sigles en anglès de compartiment per a la secreció no-convencional de proteïnes), basant-nos en la seva forma i el seu contingut. Proposem que els CUPS són l'estació de sortida per a la formació d'intermediaris vesiculars dedicats a la secreció no-convencionals que contenen Acb1.Besides conventional secretion, in which proteins are transported through the endoplasmic reticulum (ER) and the Golgi complex, unconventional secretion routes bypassing the Golgi complex have been described for different proteins in several organisms. How-ever, the mechanisms of their release remain poorly understood. It was reported that the unconventional secretion of the acyl-CoA binding protein Acb1 from Saccharomyces cerevisiae requires a diverse group of proteins including the GRASP ortholog Grh1, autophagy-related proteins, proteins involved in fusion of membranes with endosomes, members of the ESCRT-machinery, and the plasma membrane t-SNARE Sso1. How these proteins work together for Acb1 secretion remains elusive. Our findings indicate that upon nutrient starvation, the condition known to induce unconventional secretion of Acb1, Grh1 is concentrated in a phosphatidylinositol 3-kinase-dependent manner to unique membrane structures near the ER exit sites. These membranes –shaped like cups– are enriched in PI(3)P and contain the ESCRT-I protein Vps23 as well as the autophagy-related proteins Atg8 and Atg9 thereby bringing together different proteins required for Acb1 secretion. We have named these structures CUPS (Compartment for Unconventional Protein Secretion), based on their shape and content. CUPS, we propose, are the starting point for the formation of Acb1-containing vesicular intermediates dedicated for unconventional secretion.
2
Finnie, Christine. "Protein secretion by Rhizobium leguminosarum." Thesis, University of East Anglia, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361420.
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Payne, Thomas. "Protein secretion in Saccharomyces cerevisiae." Thesis, University of Nottingham, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.438772.
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De, Obeso Fernandez Del Valle Alvaro. "Protein secretion and encystation in Acanthamoeba." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31483.
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Free-living amoebae (FLA) are protists of ubiquitous distribution characterised by their changing morphology and their crawling movements. They have no common phylogenetic origin but can be found in most protist evolutionary branches. Acanthamoeba is a common FLA that can be found worldwide and is capable of infecting humans. The main disease is a life altering infection of the cornea named Acanthamoeba keratitis. Additionally, Acanthamoeba has a close relationship to bacteria. Acanthamoeba feeds on bacteria. At the same time, some bacteria have adapted to survive inside Acanthamoeba and use it as transport or protection to increase survival. When conditions are adverse, Acanthamoeba is capable of differentiating into a protective cyst. This study had three objectives. First, isolate and identify new FLA and Acanthamoeba strains. Second, identify encystation factors of Acanthamoeba. Third, identify and characterise new potential antimicrobial proteins produced by Acanthamoeba. The isolation of environmental amoebae was performed, and several strains of Acanthamoeba were identified from previously known genotypes. Also, two new species of FLA were identified: Allovahlkampfia minuta and Leptomyxa valladaresi. The dynamics of encystment were studied in different strains of Acanthamoeba. RNAseq was used to study gene expression during differentiation and identify differentially expressed genes. We identified different encystment factors including at least two encystment related proteases. A new antimicrobial zymogram was developed that identified antimicrobial proteins being secreted by Acanthamoeba. A 33 kDa protease was found to be able to lyse bacteria. We created DNA constructs encoding the protease and a lysozyme from Acanthamoeba for heterologous expression. The genes were successfully cloned. However, bacteria were not able to produce the proteins most probably due to their antimicrobial characteristics. Further studies are required regarding encystment and antimicrobial factors identified. Such experiments should help elucidate critical factors of Acanthamoeba's biology that could help treat several infections.
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Garrison, Jennifer L. "Small molecule modulation of protein secretion." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2007. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3261264.
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Ravindran, Sandeep. "Effector protein secretion by toxoplasma gondii /." May be available electronically:, 2009. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Johnson, Conkright Juliana j. "SORTING AND SECRETION OF SURFACTANT PROTEIN C." University of Cincinnati / OhioLINK, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=ucin990723467.
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Ahmad, Asma. "Protein-protein interactions in the bacterial type VI secretion system." Thesis, University of Sheffield, 2013. http://etheses.whiterose.ac.uk/4811/.
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Fu, Zhibiao. "Studies of protein secretion in escherichia coli /." View abstract or full-text, 2006. http://library.ust.hk/cgi/db/thesis.pl?BICH%202006%20FU.
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Fitchen, Nicola. "Protein localisation and secretion in Helicobacter pylori." Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273120.
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Wilson, Michael P. "Protein secretion and quorum sensing in Salmonella." Thesis, University of Newcastle Upon Tyne, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.275515.
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Srinivasan, Supriya. "Developmentally regulated protein secretion in Dictyostelium discoideum /." free to MU campus, to others for purchase, 2000. http://wwwlib.umi.com/cr/mo/fullcit?p9999318.
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Krehenbrink, Martin. "Protein secretion in Rhizobium leguminosarum biovar viciae 3841." Thesis, University of East Anglia, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432434.
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Ali, Tehmeena. "Characterisation of protein secretion systems of Actinobacillus pleuropneumoniae." Thesis, University of Nottingham, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.440122.
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Moss, Catherine Elizabeth. "G-protein coupled receptors modulating incretin hormone secretion." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648611.
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Palomäki, Tiina. "Identification of a secretion signal for the type II protein secretion pathway in Erwinia carotovora." Helsinki : University of Helsinki, 2003. http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/palomaki/.
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Nash, Kevin T. "KISS1 matastasis suppressor secretion is required for metastasis suppression." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2006. https://www.mhsl.uab.edu/dt/2008r/nash.pdf.
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Tan, Yu Pei. "The development of Lactococcus lactis as an antimicrobial agent." Thesis, Queensland University of Technology, 2010. https://eprints.qut.edu.au/39143/1/Yu_Pei_Tan_Thesis.pdf.
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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.
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Holland, Alexandria. "Optimisation of feedstock utilisation by Geobacillus thermoglucosidasius." Thesis, University of Bath, 2017. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.723323.
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Geobacillus thermoglucosidasius (GT) is a thermophilic, ethanol-producing bacterium capable of utilising both hexose and pentose sugars for fermentation. One strategy to improve fermentation yields would be to engineer GT strains to secrete hydrolases to increase the amount of available sugars from various feedstocks. Therefore, optimised protein secretion would be vital to improve feedstock utilisation. Secretion in the related mesophile Bacillus subtilis (BS) has been well studied, and several strategies have been developed to improve secretion of heterologous proteins in BS, one such strategy being the manipulation or changing of the signal peptide. One aim is to identify any differences in the secretion machinery and signal sequences between GT and BS. Another aim is to analyse any effects of overproduction of hydrolases and to identify any bottlenecks in protein secretion in GT. Using bio-informatics tools we find that although GT is a thermophile, the signal peptides in this organism do not differ significantly from those in BS. From a shotgun mass spectrometry approach it was also observed that unlike BS, GT undergoes significant cell lysis during growth releasing cytoplasmic proteins into the extracellular milieu, which could have implications on the levels of secreted hydrolases. A model enzyme was selected and over-produced at high levels in order to stress the secretion system in GT so as to identify any bottlenecks in secretion. The results thus far indicate that the rate limiting step in secretion could be post-translocation where the enzyme is degraded by proteases in the cell wall and extracellular milieu. The addition of protease inhibitor to growth media, increases the activity and abundance of the enzyme, suggesting that proteolysis may be a major factor when over-producing secreted enzymes at high levels.
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Keller, Martin. "The inflammasome : a key regulator of unconventional protein secretion /." Zürich : ETH, 2008. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17640.
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Gill, Bhupinder. "Approaches to the study of protein secretion in yeast." Thesis, University of Leicester, 1997. http://hdl.handle.net/2381/30352.
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Mutants of the yeast Saccharomyces cerevisiae that showed enhanced secretion yield of a mini-proinsulin reporter protein were isolated, following random mutagenesis, via a footprint assay screen. Three such mutants, exhibiting a 2-3 fold increase in secretion yield, were shown to contain recessive mutations in three separate chromosomal genes (ES11, ES14 and ES19). It was not possible to combine these mutations into a single strain as double heterozygous strains failed to produce viable spores. Surprisingly, all three mutants showed reduced levels of the reporter gene mRNA. Expression of a second secretory reporter, wheat -amylase, also showed altered secretory product yield and mRNA levels. However the pattern of effect was different to mini-proinsulin, indicating a specificity of action of the mutations. In contrast, cytoplasmic gene expression was unaffected, with the exception of minor codon containing heterologous genes in esi9 cells, whose expression was blocked. Generally, the three mutants appear to enhance protein secretion by improving efficiency at the initial stage of the secretory pathway probably via the co-translation mechanism. The decreased level of mRNA may result from a direct linkage to the degradation of secretory mRNA, which occurs at an increased rate in the mutants, to its mode of translation. A second set of mutants showing both enhanced and reduced secretion yields of the Aspergillus niger -galactosidase secreted protein have also been isolated. Again a random mutagenesis approach was used, but this time it was coupled to visual plate assay. These mutants await further analysis. The final part of this thesis concerns the attempted isolation of SEC gene homologues from Arabidopsis thaliana. Initially this was tried, using a functional complementation approach with sec mutants from Saccharomyces cerevisiae and two A. thaliana cDNA expression libraries but no homologues were isolated. A second approach utilising the PCR and degenerate or homologous oligonucleotide primers, designed to the SEC18 and SEC4 genes respectively, was attempted. Use of the homologous oligonucleotides led to the isolation of possible SEC4 homologous clones from one A. thaliana cDNA expression library but these clones were unable to complement a sec4 mutant. It is possible that these represent a homologue that functions in a slightly different manner in the A. thaliana secretion pathway, as the gross secretion pathway is well conserved between species.
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Tian, Ya-Min. "The involvement of protein kinase C in insulin secretion." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337551.
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Colley, Alan D. "Studies of Golgi organization and protein secretion in yeasts." Thesis, University of Edinburgh, 1995. http://hdl.handle.net/1842/19639.
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The PMR1 gene of Saccharomyces cerevisiae is predicted to encode a P-type Ca2+ ATPase (Rudolph et al., 1989). This protein has been reported as localizing to a novel Golgi-like organelle (Antebi and Fink, 1992). Consistent with Pmr1p's proposed Golgi distribution is the fact that the pleiotropic phenotype of null mutants results in defects in various Golgi processes (e.g. outer chain glycosylation, proteolytic processing, vacuolar sorting). These defects are reversible by addition of Ca2+ to the extracellular medium, supporting the proposed function as a Ca2+ pump (Rudolph et al., 1989; Antebi and Fink, 1992). Work described in this thesis was carried out to investigate the nature of the pmr1 phenotype and to further characterize the Pmr1p containing organelle(s). It is demonstrated that the pmr1 phenotype is not due to a complete bypass of the Golgi (as proposed by Rudolph et al., 1989) since Kex2p processing of a secreted protein is detected in a pmr1 mutant. There does however appear to be a change in Golgi organization in pmr1 mutants. When the organelles containing Kex2p were isolated from pmr1 and PMR1 strains the profiles of other marker enzymes recovered was significantly altered. In particular the enzyme GDPase, an early Golgi marker, colocalizes with Kex2p, a trans-Golgi network marker, in pmr1 strains. Furthermore DPAP A (Ste13p) no longer colocalizes with Kex2p in pmr1 strains. This evidence suggests that protein targeting and/or retention is altered in pmr1 strains as a result of aberrant Ca2+ homeostasis. Another possibility is that some gross reorganization of Golgi structure has occurred. These changes in enzyme localization were not reversible by addition of Ca2+ to the growth media. Pmr1p was tagged with protein A to allow isolation of organelles containing the fusion protein with IgG-Sepharose. However, after characterization of the recovered material it became clear that the fusion protein had localized to the vacuole and not the Golgi. This is probably due to the tag interfering with proper Golgi retention. This result is consistent with the vacuolar default model for membrane proteins in yeast (Roberts et al., 1992; Nothwher et al., 1993).
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Kouwen, Thijs R. H. M. "Protein secretion and disulfide bond handling in bacillus subtilis." [S.l. : [Groningen : s.n.] ; University Library Groningen] [Host], 2009. http://irs.ub.rug.nl/ppn/315686960.
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Gokoo, Suzanne. "Secretion of GBP, an infective stage-specific protein of Leishmania major." Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265838.
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Milward, Kelly. "Molecular studies using the Aspergillus nidulans #alpha#-COP homologue." Thesis, Bangor University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367360.
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Stafford, William Herbert Lee. "Analysis of the merozoite surface protein-1 complex and protein secretion in plasmodium falciparum." Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338464.
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Altman, Elliot Emr Scott. "Characterization of the SecB protein, a chaperone that facilitates protein secretion in Escherichia coli /." Diss., Pasadena, Calif. : California Institute of Technology, 1991. http://resolver.caltech.edu/CaltechETD:etd-06152007-080238.
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Pryde, James Grant. "Biogenesis of secretory granules in the bovine adrenal medulla." Thesis, University of Edinburgh, 1987. http://hdl.handle.net/1842/24238.
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Sutherland, J. L. "Studies on the patterns of secretion of extracellular proteins by Staphylococcus aureus." Thesis, University of Nottingham, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380019.
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Thwaite, Joanne E. "Factors influencing the production of Bacillus anthracis protective antigen in Bacillus subtilis." Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.369784.
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Housby, J. Nicholas. "The isolation and characterisation of conditional (Out's) and null (Out'-) secretion mutants of Erwinia carotovora subspecies carotovora (SCRI193)." Thesis, University of Warwick, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387335.
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Smith, Kenneth. "A conserved Inner Membrane Protein of Aggregatibacter actinomycetemcomitans is integral for membrane function." ScholarWorks @ UVM, 2015. http://scholarworks.uvm.edu/graddis/417.
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The cell envelope of Aggregatibacter actinomycetemcomitans, a Gram-negative pathogenic bacterium implicated in human oral and systemic disease, plays a critical role in maintenance of cellular homeostasis, resistance to external stress, and host'pathogen interactions. Our laboratory has identified a novel gene product, morphogenesis protein C (MorC), deletion of which leads to multiple pleotropic effects pertaining to membrane structure and function. The MorC sequence was determined to be conserved in Gammaproteobacteria. Based on this bioinformatic analysis, the functional conservation of this protein was investigated utilizing an A. actinomycetemcomitans morC mutant as a model system to express homologs from four phylogenetically diverse representatives of the Gammaproteobacteria: Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis. MorC from all organisms restored at least one of the A. actinomycetemcomitans mutant phenotypes, implying that the protein is functionally conserved across Gammaproteobacteria. Further, deletion mutagenesis indicated that the last 10 amino acids of the carboxyl terminus were necessary to maintain the integrity of the membrane. The observed pleiotropic effects suggested alterations in the membrane protein composition of the morC mutant. Stable isotope dimethyl labeling in conjunction with mass spectrometry was employed to quantitatively determine the differences in the abundance of membrane proteins of the isogenic mutant and wild-type strains. A total of 665 envelope associated proteins were identified and functionally annotated using bioinformatic tools. All proteins, except MorC, were detected in the mutant strain. However, 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain. These proteins were ascribed functions associated with protein quality control, oxidative stress response, and protein secretion systems.
One protein found to be reduced was a component of the fimbrial secretion system of A. actinomycetemcomitans. The significance of this finding was unclear due to the afimbriated nature of the laboratory strain used in the study. Therefore, the defect in fimbriation was identified and complemented in trans. The transformed strain displayed all of the hallmarks of a naturally fimbriated strain including: a distinct star-like colony morphology; robust biofilm formation; and presence of fimbriae as detected by electron microscopy. The isogenic morC mutant strain transformed with an identical plasmid did not display any fimbriated phenotypes. The role of MorC in fimbriae production of a naturally fimbriated strain was investigated by inactivation of morC in a clinical isolate. The mutant strain displayed phenotypes typically associated with inactivation of morC. However, fimbriae were still observed on the surface, although in lesser amounts on some individual bacteria, and this strain formed a biofilm with volume similar to the parent. Interestingly, significant changes in microcolony architecture of the biofilm were observed by confocal microscopy.
MorC plays a critical role in maintaining secretion of major virulence determinants of A. actinomycetemcomitans. Specific changes in the protein composition of the cell envelope indicate a direct or compensatory role of these proteins in maintaining membrane physiology. The functional conservation of MorC also implies an important role for this protein in other Gram-negative bacteria. This work suggests a role of MorC as an accessory or a scaffold protein involved in secretion.
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Kornacker, Michael Gilbert. "Analysis of pullulanase secretion from Klebsiella pneumoniae strain K21." Thesis, University of Leicester, 1988. http://hdl.handle.net/2381/34437.
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Strains of the Gram-negative bacterium Klebsiella pneumoniae secrete pullulanase, a maltose-inducible starch debranching enzyme that exists as a cell surface bound intermediate. Three classes of secretion mutants were obtained by transposon In 10 mutagenesis. Class I and class III mutants carry Tn10 insertions in pullulanase secretion genes. Class II mutants carry insertions in regulatory loci that are required for the high level expression of pullulanase and other maltose-inducible genes (the maltose regulon). One such locus appears to correspond to a previously unknown locus. The phenotypes of the secretion mutants and the analysis of E.coli expressing pullulanase and/or cloned pullulanase secretion genes suggest that pullulanase secretion functions are involved in translocating pullulanase across the outer membrane and in releasing it from the cell surface. Most if not all pullulanase secretion genes are located to both sides of the structural gene for pullulanase (pu1A). Pullulanase was found to be a lipoprotein. Surprisingly, secreted pullulanase also carried lipid. However, strain K21 differed from other strains of Klebsiella pneumoniae by its ability to secrete not only acylated pullulanase but also a second, unacylated form of pullulanase. Strain K21 is also unusual because of its ability to secrete most pullulanase during logarithmic growth. This pullulanase corresponds to the unacylated pullulanase, with the remaining secreted pullulanase being acylated and secreted during stationary phase, as is the case for most or all pullulanase of other strains of Klebsiella pneumoniae. Strain K21 is also unusual because of the high level expression of the maltose regulon, including pulA, in the absence of maltose. This property, including the unusual features described above, may be a consequence of the selection of strain K21 for high level commercial production of pullulanase. Models for pullulanase secretion are discussed and approaches towards increasing the efficiency of commercial pullulanase production by strain K21 are outlined.
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Entwistle, Joanna. "Isolation of genes involved in protein secretion in Aspergillus niger." Thesis, University of Nottingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.311669.
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Huppert, Laura Ann. "Characterization of the Bacillus subtilis ESX-Type Protein Secretion System." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:15821590.
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Esat-6 Protein Secretion Systems are required for the virulence of several human pathogens, most notably Mycobacterium tuberculosis and Staphylococcus aureus. Gene clusters coding for ESX systems have been identified amongst many organisms including the highly tractable non-pathogenic bacterium Bacillus subtilis. In this work, I develop a model system in B. subtilis to study ESX-type Secretion Systems. First, I demonstrate that the B. subtilis yuk/yue locus encodes a functional secretion system. Then, I utilize this system to study the mechanism of protein export. I show that only the N-terminal ATPase domain in the conserved ATPase is essential for substrate export and demonstrate the first evidence of secretion of an intact dimeric complex that requires a composite recognition signal formed by both members of the complex. I also study the function of the B. subtilis ESX system and find that certain yuk mutants have a biofilm phenotype. Finally, I find that the yuk/yue operon is upregulated in the B. subtilis undomesticated strain 3610 and find that none of the Yuk proteins are required for substrate export in this background. Together, these findings help to elucidate the mechanism and function of this important class of secretion systems.
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Yan, J. "Mass spectrometric studies of proteins and protein complexes involved in bacterial secretion and regulatory systems." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1426517/.
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This paper presents the results of a large scale empirical study of coherent dependence clusters. All statements in a coherent dependence cluster depend upon the same set of statements and affect the same set of statements; a coherent cluster's statements have ‘coherent’ shared backward and forward dependence. We introduce an approximation to efficiently locate coherent clusters and show that it has a minimum precision of 97.76%. Our empirical study also finds that, despite their tight coherence constraints, coherent dependence clusters are in abundance: 23 of the 30 programs studied have coherent clusters that contain at least 10% of the whole program. Studying patterns of clustering in these programs reveals that most programs contain multiple substantial coherent clusters. A series of subsequent case studies uncover that all clusters of significant size map to a logical functionality and correspond to a program structure. For example, we show that for the program acct, the top five coherent clusters all map to specific, yet otherwise non-obvious, functionality. Cluster visualization also brings out subtle deficiencies in program structure and identifies potential refactoring candidates. A study of inter-cluster dependence is used to highlight how coherent clusters are connected to each other, revealing higher-level structures, which can be used in reverse engineering. Finally, studies are presented to illustrate how clusters are not correlated with program faults as they remain stable during most system evolution.
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Maldonado, Rodrigo. "Erythropoietin provides a molecular model for protein burden during intracellular processing and secretion of recombinant proteins." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/erythropoietin-provides-a-molecular-model-for-protein-burden-during-intracellular-processing-and-secretion-of-recombinant-proteins(79ca36c2-75f2-4426-8ab9-d74994fa8ed9).html.
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Several elements of the unfolded protein response (UPR) signaling pathway have been engineered in Chinese hamster ovary (CHO) cells with variable effect on recombinant protein (RP) production. To gain insight into the relationship between cell productivity and UPR activation, two recombinant CHO cell lines secreting different amounts (5-fold difference) of erythropoietin (EPO) were characterised in terms of growth, RP production, intracellular EPO content and UPR activation. Differential productivity was correlated with the intracellular amounts of EPO, without affecting cell growth or expression of UPR markers. We found that intracellular EPO presents a different glycosylation status than secreted EPO, representing a possible bottleneck for EPO secretion. To gain insight into the UPR activation during batch culture, the high producing CHO-EPO cell line was used to evaluate UPR target gene expression during batch culture and tunicamycin (Tm)-induced ER stress. Increased abundance of mRNAs related to UPR activation was observed during late days of batch culture of CHO-EPO cells, suggesting UPR activation during batch culture is a consequence of environment changes during batch culture rather than RP expression. Induction of ER stress by the addition of Tm produced changes in the abundance of the UPR-related mRNAs in a pattern similar to that seen during batch culture. mRNA targets associated with the UPR that had not been previously described in the context of RP production in CHO cells were identified (such as ODZ4, HERPUD1 and SQSTM1). Finally, cells expressing EPO under the control of an inducible promoter were used to study the effect of recombinant protein expression on cell phenotype, focusing on the UPR and cell metabolism. Induction of EPO expression was not able to activate the UPR or affect cell growth. Instead, changes in the abundance of metabolites associated with the control of shuttle systems that transport electrons across the mitochondrial membrane were observed in induced cells. These shuttle systems are involved in the transport and conversion of glycerol, citrate, isocitrate and malate, which collectively regulate the redox balance of the cell. In summary, I did not find any evidence of UPR activation on response to RP expression, possible because EPO is not a good inducer of stress compared to other proteins, or I did not reached the amounts of production needed to stress the cell. Despite this, I found metabolic changes associated with the induction of RP expression. Further work will focus on characterisation if this metabolic response is something general to RP induction or if it is a protein-specific characteristic.
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Alzahrani, Ashwag. "Identification of Human Proteins Interacting with the Protein IcsB of Shigella flexneri." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/38333.
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Wei, Peter. "The structural characterization of the Saccharomyces cerevisiae alpha mating factor secretion signal for recombinant protein secretion in Pichia pastoris." Scholarly Commons, 2015. https://scholarlycommons.pacific.edu/uop_etds/177.
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The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation with rapid growth to high cell densities and provides complex posttranslational modifications. The most successful and commonly used secretion signal leader in Pichia pastoris has been the MAT α prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently even with the MAT α prepro secretion signal. Some strategies to enhance secretion efficiency involved modifying the secretion signal leader. Based on the knob-socket model and Jpred3 ( a secondary structure predictor), eleven deletions of MAT α prepro secretion signal and one MAT α pre double pro-peptide mutant was engineered and assayed with either horseradish peroxidase (HRP), or Candida antarctica lipase B reporter protein to evaluate the correlation between secondary structure and secretion level. In addition, structural analysis through circular dichroism was completed for the wild type pro-peptide and a mutant pro-peptide to evaluate differences in secondary structure. Results suggest pro-peptide amino acids 75-78 play an important role in determining secretion level and a higher secretion level tends to associate with secondary structures that are less defined. With these analyses, optimization of secretion systems can be achieved to impact the fields of science, industry, healthcare, and economics worldwide.
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Ghanem, Simona S. "A Role for CEACAM2 protein in Insulin Secretion, Clearance and Action." University of Toledo Health Science Campus / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=mco1491497714470047.
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Sibanda, Ntsako. "Evaluation of high recombinant protein secretion phenotype of saccharomyces cerevisiae segregant." Thesis, University of Limpopo, 2016. http://hdl.handle.net/10386/1803.
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Thesis (MSc. (Biochemistry)) --University of Limpopo, 2016The ever increasing cost of fossil-based fuels and the accompanying concerns about their impact on the environment is driving research towards clean and renewable sources of energy. Bioethanol has the potential to be a replacement for liquid transportation fuels. In addition to its near zero nett carbon dioxide emissions, bio-ethanol has a high energy to weight ratio and can easily be stored in high volumes. To produce bioethanol at economically competitive prices, the major cost in the production process needs to be addressed. The addition of enzymes to hydrolyse the lignocellulosic fraction of the agricultural waste to simple sugars is considered to be the major contributor to high production cost. A consolidated bioprocess (CBP) which ideally combines all the steps that are currently accomplished in different reactors by different microorganisms into a single process step would be a more economically feasible solution. In this study the potential of yeast hybridization with a CBP approach was used. In order to evaluate the reduction or elimination of the addition of cellulolytic and hemi-cellulolytic enzymes to the ethanol production process. High cellobiohydrolase I secreting progeny from hybridization of an industrial bioethanol yeast strain, S. cerevisiae M0341, and a laboratory strain S. cerevisiae Y294 were isolated. In order to determine if this characteristic was specific to cellobiohydrolase I secretion, these strains were evaluated for their ability to secrete other relevant recombinant hydrolase enzymes for CBP-based ethanol production. A total of seven S. cerevisiae strains were chosen from a progeny pool of 28 supersecreting hybrids and reconstructed to create two parental strains; S. cerevisiae M0341 and S. cerevisiae Y294, together with their hybrid segregants strains H3M1, H3M28, H3H29, H3K27 and H3O23. Three episomal plasmids namely pNS201, pNS202 and pNS203 were constructed; these plasmids together with two already available plasmids, namely pRDH166 and pRDH182 contained genes for different reporter enzymes, namely β-glucosidase I, xylanase II, endoglucanase lll, cellobiohydrolase l and α-glucuronidase. To allow for selection of the episomal plasmids, homologous recombination was used to replace the functional URA3 gene of selected strains, with the non-functional ura3 allele from the Y294 strain. Enzyme activity was used as an indicator of the amount of enzyme secreted. Fermentation studies in a bioreactor were used to determine the metabolic burden imposed on the segregants expressing the cellobiohydrolase at high levels. In addition all segregants were tested for resistance to inhibitors commonly found in pre-treated lignocellulosic material. The M28_Cel7A was found to be the best secretor of Cel7A (Cellobiohydrolase l); however it seems as though this phenomenon imposes a significant metabolic burden on the yeast. The supersecreting hybrid strains cannot tolerate lignocellulosic inhibitors at concentrations commonly produced during pretreatment
The National Research Foundation - Renewable Energy Scholarship (NRF-RSES)
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Komar, Joanna. "Activity of the holo-translocon and its individual components in protein secretion and membrane protein insertion." Thesis, University of Bristol, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.702120.
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The ubiquitous Sec machinery facilitates protein movement across or integration of proteins into the cytoplasmic membrane in a post- or cotranslational manner, respectively. In vivo, the bacterial core SecYEG translocon associates with additional membrane components, SecDF-YajC and YidC, forming a complex referred to as the holo-translocon (HTL). A recent breakthrough came from the isolation of a stable complex comprising all seven subunits, enabling analysis of HTL function and interactions between its constituents. HTL's activity in protein secretion and membrane protein insertion was analysed both in the presence and absence of the proton-motive force (PM F). Findings presented here suggest that the HTL supports both processes. Interestingly, it appears less efficient in protein secretion than SecYEG alone, but more responsive to the PMF. Nevertheless, the HTL is more effective at membrane protein incorporation for the majority of substrates analysed in this study. It also appears more efficient at assembly of membrane protein complexes investigated here. These findings suggest that the HTL complex is preferential for membrane protein insertion, whereas SecYEG is more effective in protein secretion. An activated conformation of SecYEG was also investigated in this study. For this purpose, cross-links were designed within the channel based on the structure representing its unlocked state when bound to a signal sequence. Fixing the channel in two different conformations, representing unlocking of the SecY channel and displacement of the plug domain, resulted in its increased activity in protein secretion. However, only the latter conformation had an effect on membrane protein insertion, which suggests major differences in the activation mechanism between these processes. Findings presented here have helped in the understanding of the recent structure of the HTL. This structural information together with functional studies reported here address unknown aspects of the fundamental problem in biology: membrane protein insertion, folding and assembly.
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Upritchard, Hamish Graeme, and n/a. "Host interactions with Pseudomonas aeruginosa : a proteomic approach." University of Otago. Department of Biochemistry, 2005. http://adt.otago.ac.nz./public/adt-NZDU20060804.101030.
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Pseudomonas aeruginosa is an opportunistic bacterial pathogen associated with severe nosocomial infections in immunocompromised hosts and patients with cystic fibrosis (CF). During infection the bacteria secrete proteins that are essential to the infection process. Several of these virulence-associated proteins have been identified using genetic methods. The aim of this research, using a proteomic approach, was to identify novel extracellular proteins that are secreted by P. aeruginosa during infection of a CF patient.
Extracellular proteins from P. aeruginosa strain PAO1 grown in vitro were separated by two-dimensional gel electrophoresis (2-DE). The humoral response of chronically infected CF patients to the separated proteins was elucidated using western blotting. Growth phase, cell population and iron limitation were identified as important regulators of the extracellular proteome. The number of extracellular proteins significantly increased upon entry into stationary phase, as did the number of proteins detected by CF patient sera. The detection of several known quorum-controlled proteins by patient sera indicated the importance of this regulatory mechanism during infection. In iron-limiting medium, the proportion of proteins detected by CF patient sera significantly increased compared to extracellular proteins from cells grown in iron-replete conditions. Proteomic analysis of a PAO1 pvdS mutant strain showed that PvdS (an iron-regulated alternative sigma factor) directs production of many extracellular proteins made during infection of a CF patient. Examination of extracellular proteins from a second strain, PA4, indicated it had a shared set of extracellular proteins. The identities of selected proteins were determined and these included well-characterised extracellular virulence factors such as elastase (LasB). Also identified were proteins with a potential virulence role such as azurin (a copper containing redox protein), PA2939 (a likely aminopeptidase) and proteins with unknown functions. This study provides the first evidence for the production of these proteins during infection. In summary, the proteomics methodology developed here facilitated the rapid identification and enumeration of proteins secreted by P. aeruginosa during infection.
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Li, Zhiguo. "Structure, secretion, and proteolysis study of MBP-containing heterologous proteins in Pichia pastoris." Scholarly Commons, 2010. https://scholarlycommons.pacific.edu/uop_etds/2415.
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The E. coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N -terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris , a popular eukaryotic host for heterologous protein expression. When MBP was fused as an N -terminal partner to several C -terminal cargo proteins expressed in this yeast, proteolysis occurred between the two peptides, and MBP reached the extracellular region unattached to its cargo. However, in two of three instances, the cargo protein reached the extracellular region as well, and its initial attachment to MBP enhanced its secretion from the cell. Extensive mutagenesis of the spacer region between MBP and its C -terminal cargo protein could not inhibit the cleavage although it did cause changes in the protease target sites in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that an uncharacterized P. pastoris protease attacked at different locations in the region C -terminal of the MBP domain, including the spacer and cargo regions, but the MEP domain could still act to enhance the secretion of certain cargo proteins. The attempt to identify the unknown protease was unsuccessful. However, in contrast to other fusion partners, MBP was secreted with the cargo when it was fused as a C -terminal peptide to an N -terminal cargo protein. These studies provide insights into the role of proteases and fusion partners in the secretory mechanism of P. pastoris , suggesting new strategies to optimize this expression system.
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Guo, Dongni Lily Centre for Vascular Research Faculty of Medicine UNSW. "Protein kinase A and related pathways in the regulation of apolipoprotein E secretion and catalase activity." Awarded By:University of New South Wales. Centre for Vascular Research, 2009. http://handle.unsw.edu.au/1959.4/41512.
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Cyclic-AMP dependent protein kinase A (PKA) regulates traffic of multiple proteins at different stages along the constitutive secretory pathway. PKA effects are regulated by protein phosphatases, which reverse the actions of PKA by dephosphorylation of PKA-substrates. Localization of specific PKA effects is mediated by the binding of A-kinase anchoring proteins (AKAPs). Apolipoprotein E (apoE) is an important regulator of lipid metabolism and atherosclerosis, and represents a large proportion of total protein constitutively secreted from macrophages. The signalling and trafficking pathways regulating secretion of apoE are unknown. Catalase is a peroxisomal enzyme which contributes to defence against hydrogen peroxide (H2O2). The primary hypothesis of this thesis is PKA and related protein phosphatase pathways are involved in the regulation of apoE secretion. The secondary hypothesis is that these pathways also regulate cellular clearance of H2O2. In Chapter Three, I have investigated the role of PKA in apoE secretion from primary human macrophages. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI14-22) all decreased basal secretion of apoE by between 50-80% whereas apoE mRNA or cellular protein are unaffected. Disruption of PKA-AKAP anchoring also significantly inhibited apoE secretion from human macrophages. Secretion of apoE was not immediately stimulated by PKA activity, suggesting that although PKA activity may be permissive for apoE secretion, it is in itself insufficient to stimulate apoE secretion above basal levels. Data from confocal microscopy and live cell imaging revealed PKA inhibition paralysed apoE vesicular movement from and to the plasma membrane. In Chapter Four, I investigated the effects of protein phosphatase 2B (PP2B) inhibition on apoE secretion by cyclosporin A (CsA). This was found to dose- and time-dependently inhibit secretion of apoE from primary human macrophages and increased cellular accumulation of apoE without affecting apoE mRNA levels. The role of PP2B in regulating apoE secretion was confirmed by using additional peptide and chemical inhibitors of PP2B. This effect was independent of the known inhibition of ABCA1 by CsA. Live cell imaging and confocal microscopy all demonstrated that inhibition of PP2B did not affect the apparent cellular distribution of apoE. Biochemical and microscopy studies indicated distinct mechanisms for PKA and PP2B regulation of apoE secretion. Chapter Five identified PKA-anchoring AKAPs in human macrophages, and investigated AKAP220 expression and its role in PKA-dependent processes relevant to atherosclerosis. AKAP220 protein was absent in human monocytes but was detectable after their differentiation into macrophages, with stable expression during late stages of maturation. It was also present in Chinese Hamster Ovary cells (CHO) cells. AKAP220 silencing had no effects on lipoprotein cholesteryl ester accumulation, total cellular apoE levels, apoE secretion or cholesterol efflux from human macrophages. Confocal microscopy in CHO cells revealed peroxisomal localisation of AKAP220. Catalase activity was confirmed to be PKA-regulated process, and AKAP220 was found to be a negative regulator of catalase activity, such that cell lysate catalase activity increased during AKAP220 silencing. AKAP220 silencing also decreased basal secretion of H2O2, detected using a sensitive and specific Amplex?? Red assay kit from intact CHO monolayers. In conclusion, this thesis has provided evidence that apoE secretion occurs via PKA- and PP2B-dependent pathways in human macrophages, and has identified the A-kinase anchoring protein AKAP220 as a regulator of cellular H2O2 clearance. These results will provide a basis for future investigations into the roles of PKA-related pathways in apoE secretion and catalase activity.
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Chen, Chen. "Studies on Selective Protein Loading onto Extracellular Membrane Vesicles of a Novel Cold-Adapted Bacterium, Shewanella vesiculosa HM13." Kyoto University, 2020. http://hdl.handle.net/2433/253331.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第22495号
農博第2399号
新制||農||1076(附属図書館)
学位論文||R2||N5275(農学部図書室)
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 栗原 達夫, 教授 小川 順, 教授 木岡 紀幸
学位規則第4条第1項該当
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Miller, Paul E. "Differential secretion from prestored heterogeneous protein sources is the basis of regulated nonparallel digestive enzyme secretion by the exocrine pancreas." Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74316.
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For two decades, multiple observations of nonparallel pancreatic secretion, wherein digestive enzyme proportions change rapidly following various digestive stimuli, have conflicted with the concept of exocrine pancreatic homogeneity and the exocytosis model of parallel synthesis, transport and secretion of proteins. Evidence of pancreatic heterogeneity is presented, potentially resolving this longstanding controversy. Correlation and regression analysis simultaneously demonstrated exocytosis and nonparallel secretion, suggesting the existence of multiple heterogeneous exocytotic pathways. Next, heterogeneous prestored pancreatic protein sources were directly demonstrated using double isotopic labelling; temporal and secretagogue-specific regulation of the heterogeneous secretory sources was uncovered. Finally, specific enzyme proportions were linked to the heterogeneous sources by densitometric measurements of electrophoretic gels of secreted proteins. Thus, it appears that differential secretion from heterogeneous sources of prestored secretory proteins containing unique proportions of digestive enzymes is the basis of regulated nonparallel secretion in the exocrine pancreas.
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Kriel, Johan Hendrik. "Development of synthetic signal sequences for heterologous protein secretion from Saccharomyces cerevisiae." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/53364.
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Thesis (MSc)--Stellenbosch University, 2003.ENGLISH ABSTRACT: Protein secretion and intracellular transport are highly regulated processes and involve the interplay of a multitude of proteins. A unique collection of thermosensitive secretory mutants allowed scientists to demonstrate that the secretory pathway of the yeast Saccharomyces cerevisiae is very similar to that of the higher eukaryotes. All proteins commence their journey in the endoplasmic reticulum, where they undergo amino-linked core glycosyl modification. After passage through the Golgi apparatus, where the remodelling of the glycosyl chains is completed, proteins are transported to their final destinations, which are either the cell surface, periplasmic space or the vacuole. Proteins destined for secretion are usually synthesised with a transient amino-terminal secretion leader of varying length and hydrophobicity, which plays a crucial role in the targeting and translocation of their protein cargo. Considerable effort has been made to elucidate the molecular mechanisms involved in these processes, especially due to their relevance in a rapidly expanding biotech industry. The advantages of S. cerevisiae as a host for the expression of recombinant proteins are well documented. Unfortunately, S. cerevisiae is also subject to a number of drawbacks, with a relative low product yield being one of the major disadvantages. Bearing this in mind, different secretion leaders were compared with the aim of improving the secretion of the LKA 1 and LKA2 a-amylase enzymes from the S. cerevisiae secretion system. The yeast Lipomyces kononenkoae is well known for its ability to degrade raw starch and an improved secretion of its amylase enzymes from S. cerevisiae paves the way for a potential one-step starch utilisation process. Three sets of constructs were prepared containing the LKA 1 and LKA2 genes separately under secretory direction of either their native secretion leader, the S. cerevisiae mating pheromone a-factor (MFa1) secretion leader, or the MFa1 secretion leader containing a synthetic C-terminal spacer peptide (EEGEPK). The inclusion of a spacer peptide in the latter set of constructs ensured improved Kex2p proteolytic processing of the leader/protein fusion. Strains expressing the amylase genes under their native secretion leaders resulted in the highest saccharolytic activity in the culture medium. In contrast to this, strains utilising the synthetic secretion leader produced the highest fermentation yield, but had a lower than expected extracellular activity. We hypothesise that the native amylase leaders may function as intramolecular chaperones in the folding and processing of their passenger proteins, thereby increasing processing efficiency and concomitant enzyme activity.
AFRIKAANSE OPSOMMING: Proteïensekresie en intrasellulêre transport is hoogs gereguleerde prosesse en betrek die onderlinge wisselwerking van 'n verskeidenheid proteïene. 'n Unieke versameling van temperatuur-sensitiewe sekresiemutante het wetenskaplikes in staat gestelom die ooreenkoms tussen die sekresiepad van die gis Saccharomyces cerevisiae en dié van komplekser eukariote aan te toon. Alle proteïene begin hul reis in die endoplasmiese retikulum, waartydens hulle ook amino-gekoppelde kernglikosielveranderings ondergaan. Nadat die proteïene deur die Golgi-apparaat beweeg het, waar die laaste veranderings aan die glikosielkettings plaasvind, word hulle na hul finale bestemmings, waaronder die seloppervlak, die periplasmiese ruimte of die vakuool, vervoer. Proteïene wat vir sekresie bestem is, word gewoonlik met 'n tydelike, amino-eindpuntsekresiesein, wat 'n kritiese rol in die teiken en translokasie van hul proteïenvrag speel, gesintetiseer. Heelwat pogings is in hierdie studie aangewend om die molekulêre meganismes betrokke by hierdie prosesse te ontrafel, veral as gevolg van hul toepaslikheid in 'n vinnig groeiende biotegnologiebedryf. Die voordele van S. cerevisiae as 'n gasheer vir die uitdruk van rekombinante proteïene is alombekend. S. cerevisiae het egter ook verskeie nadele, waaronder die relatiewe lae produkopbrengs die belangrikste is. Teen hierdie agtergrond, is verskillende sekresieseine met mekaar vergelyk met die doelom die sekresie van die LKA 1 en LKA2 a-amilasegene vanuit die S. cerevisiae-uitdrukkingsisteem te verbeter. Die gis Lipomyces kononenkoae is bekend vir sy vermoeë om rou stysel af te breek en 'n verbeterde sekresie van sy amilasegene vanuit S. cerevisiae baan die weg vir 'n moontlike een-stap styselgebruiksproses. Drie stelle konstrukte is gemaak wat die LKA 1- en LKA2- gene onafhanklik onder sekresiebeheer van onderskeidelik hul inheemse sekresiesein, die S. cerevisiae paringsferomoonsekresiesein (MFa1) of die MFa1-sekresiesein met 'n sintetiese koppelingspeptied aan die C-eindpunt (EEGEPK), plaas. Die insluiting van 'n koppelingspeptied in die laasgenoemde stel konstrukte verseker verbeterde Kex2p proteolitiese prosessering van die sein/proteïenfusie. Rasse wat die amilasegene onder beheer van hul inheemse sekresieseine uitdruk, het die beste saccharolitiese aktiwiteit in die kultuurmedia getoon. In teenstelling hiermee, het rasse wat van die sintetiese sekresiesein gebruik maak, die beste fermentasie-opbrengs getoon, maar met 'n laer as verwagte ekstrasellulêre aktiwiteit. Ons vermoed dat die inheemse amilaseseine as intramolekulêre begeleiers optree in die vou en prosessering van hul proteïenpassasiers, wat lei tot verbeterde prosessering en ensiemaktiwiteit.
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Creasey, Elizabeth Anne. "Protein interactions in the Type III secretion system of Enteropathogenic Escherichia Coli." Thesis, Imperial College London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.410342.
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