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1
Baas, Tracey Lynn. « The design, synthesis, and characterization of template assembled synthetic proteins / ». Thesis, Connect to this title online ; UW restricted, 2000. http://hdl.handle.net/1773/11561.
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2
Draffan, Lynda Catherine. « Chemical synthesis of proteins ». Thesis, University of Edinburgh, 1996. http://hdl.handle.net/1842/13715.
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Morton, Gail Helen. « The chemical synthesis of proteins ». Thesis, University of Edinburgh, 1997. http://hdl.handle.net/1842/15437.
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The viability of extending the present methodology designed for the solid phase synthesis of peptides has been investigated. Using the catalytic domain of stromelysin (SCD. 173 residues) as the model system, a number of different factors affecting the preparation and purification of chemically synthesised proteins are examined. The purification SCD, prepared using stepwise solid phase synthesis is described. Following characterisation, it is evident that protein of the correct primary sequence has been prepared, furthermore, preliminary studies centred on the enzymatic activity of SCD indicate that active protease has been isolated. However, comparison of the conformational and biophysical properties of chemically synthesised SCD with the recombinant counterpart, suggests that there are problems associated with the folding of the synthetic SCD. The construction of chemically synthesised SCD via convergent protein synthesis is also described. Two different coupling strategies involving classical and azide fragment condensation are examined where it has been highlighted that the overall success of each fragment coupling is greatly dependent on the peptide length and sequence. As well as comparing methods for the preparation and coupling of fully and minimally protected peptides, general procedures for both solution and solid phase fragment coupling are discussed. A novel strategy for the convergent synthesis of peptides and proteins has been investigated. In this total chemical synthesis, two minimally protected peptides are joined through unique, mutually reactive functional groups, yielding a peptide analogue with a thioether replacement for the native peptide bond at the site of ligation. A general route to C-terminal sulfhydryl and N-terminal haloacetylated peptides is presented, accompanied with results of the preliminary ligation studies.
4
Walker, Douglas Gordon. « Characterization of immediate-early and early proteins of murine cytomegalovirus synthesized in permissive and nonpermissive cells ». Thesis, University of British Columbia, 1985. http://hdl.handle.net/2429/25985.
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The gene products produced by murine cytomegalovirus (MCMV) in infected cells prior to viral DNA synthesis are believed to control the interaction of the virus with the cells, determining whether a permissive infection results, with virus replication, or whether further virus gene expression is inhibited, resulting in a latent or abortive infection. The aim of this study was to characterize the early viral gene products that are produced in permissive and nonpermissive cells.
The proteins produced in 3T3-L1 cells, permissively infected with MCMV, during the first six hours of infection (the period prior to viral DNA replication) were characterized by polyacrylamide gel electrophoresis. Ten of the proteins were classified as immediate-early (IE) and seven as early according to their time of synthesis and also according to their synthesis in the presence of actinomycin D following the reversal of a cycloheximide mediated block in protein synthesis. The estimated molecular weights ranged from 28K - 100K. The synthesis of a dominant IE protein of 100K was significantly increased, after the reversal of a cycloheximide block, compared to unenhanced conditions. The synthesis of two other major IE proteins of 96K and 89K were also significantly enhanced by this treatment. The 100K and 89K proteins partitioned with the nuclear, cytoplasmic and cytoskeletal fractions, while the 96K protein partitioned more strongly with the nuclei. These proteins were phosphorylated. The other IE proteins were synthesized in lesser amounts. The major early proteins, which had molecular weights of 39K and 36K, were also phosphorylated and were exclusively nucleus-associated. A number of the IE and early proteins had affinity for native and denatured DNA-cellulose.
The same major IE and early proteins were identified in nonpermissively infected J774A.1 macrophage cells. Although 0.6% of these cells became permissively infected with MCMV and the rest appeared to be nonpermissively infected, viral DNA and late protein synthesis was not detected. The major difference between the proteins produced in 3T3-L1 cells and J774A.1 cells was the affinity of the 96K protein for denatured DNA-cellulose, which was only observed when the protein was synthesized in J774A.1 cells.
The main IE and early MCMV induced proteins were also synthesized in nonpermissively infected human fibroblast cells. The only difference between the proteins produced in these cells and 3T3-L1 cells was that the 100K IE protein appeared to have a greater nuclear-affinity, when produced in the human fibroblasts, than was found when synthesized in infected 3T3-L1 cells.
In conclusion, a larger number of IE and early MCMV-induced proteins were identified in infected cells than had been previously characterized. There was no evidence of restricted MCMV gene expression occurring in two different cell types that were nonpermissively infected. This appeared to indicate that, in the nonpermissive experiments described, MCMV replication was inhibited at the stage of viral DNA synthesis.Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate
5
Scott, Felicia Yi Xia. « Controlled Hybrid Material Synthesis using Synthetic Biology ». Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/86147.
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The concept of creating a hybrid material is motivated by the development of an improved product with acquired properties by amalgamation of components with specific desirable traits. These new attributes can range from improvements upon existing properties, such as strength and durability, to the acquisition of new abilities, such as magnetism and conductivity. Currently, the concept of an organic-inorganic hybrid material typically describes the integration of an inorganic polymer with organically derived proteins. By building on this idea and applying the advanced technologies available today, it is possible to combine living and nonliving components to synthesize functional materials possessing unique abilities of living cells such as self-healing, evolvability, and adaptability. Furthermore, artificial gene regulation, achievable through synthetic biology, allows for an additional dimension of the control of hybrid material function.
Here, I genetically engineer E. coli with a tightly controlled artificial protein construct, allowing for inducible expression of different amounts of the surface anchored protein by addition of varying concentrations of L-arabinose. The presence of the surface protein allows the cells to bind nonliving nanoparticle substrates, effectively turning the cells into living crosslinkers. By using the living crosslinker, I was able to successfully synthesize a robust, macroscale living-nonliving hybrid material with magnetic characteristics. Furthermore, by varying the particle size and inducer concentration, the resulting material exhibited alterations in structure and function. Finally, I was able to manipulate material kinetics within a PDMS channel by applying fluctuating magnetic fields and demonstrate material durability. These results demonstrate the ability to manipulate synthesis of living-nonliving hybrid materials, which demonstrate the potential for use in promising applications in areas such as environmental monitoring and micromachining. Additionally, this work serves as a foundational step toward the integration of synthetic biology with tissue engineering by exploiting the possibility of controlling material properties with genetic engineering.Ph. D.
6
Schwartz, Anne. « Characterization of normal and androgen resistant-genital skin fibroblasts using high-resolution two-dimensional gel electrophoresis ». Thesis, McGill University, 1985. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=63378.
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Zhang, Yinfeng, et 张银凤. « Protein chemical synthesis by serine and threonine ligation ». Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/202359.
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Landmark advances in the field of synthetic protein chemistry have enabled the preparation of complex, homogeneous proteins, including those that carry specific posttranslational modifications (PTMs). In addition, chemical synthesis will allow one to incorporate unnatural elements to generate new biologics with altered properties and functions. Native chemical ligation (NCL) is a milestone in the chemical synthesis of proteins (Kent et al., Science, 1994, 266, 776-779), in which a C-terminal peptide thioester and an N-terminal cysteine (Cys)-containing peptide-both in side-chain unprotected forms-are selectively coupled to generate a natural peptidic linkage at the site of ligation. This method requires a cysteine at the optimal convergent ligation site. However, Cys is one of the least abundant amino acids in natural proteins. Therefore, the development of new ligation methods at other amino acids will be necessary and important in this regard.
Along these lines, our laboratory has developed a novel thiol-independent approach-serine/threonine ligation (STL). It uses the N-terminal serine or threonine of a peptide segment to chemoselectively react with another peptide segment with a C-terminal salicylaldehyde ester to form an N,O-benzylidene acetal linked product, followed by acidolysis to afford the final product at the natural Ser/Thr site. To extend the application of STL in chemical protein synthesis, we have developed a robust method for the preparation of peptide salicylaldehyde esters via Fmoc-based solid phase peptide synthesis. Furthermore, we have successfully applied this ligation method in the convergent synthesis of peptide drugs of significant therapeutic importance, including Teriparatide (Forteo), Corticorelin (oCRH), Exenatide (Byetta) and Tesamorelin (hGHRH). Of significance, we have demonstrated the effectiveness of our STL in the assembly of a more complex target of biological interest: human erythrocyte acylphosphatase (~ 11 kDa).
In summary, we have developed a new serine/threonine ligation, which can be effectively used to synthesize peptides and proteins. As there are countless serine and threonine residues in natural proteins, particularly those carrying posttranslational modifications, this method is anticipated to offer new opportunities in synthetic protein chemistry and chemical biology.published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy
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Johnston, Julie Catherine. « In vitro translation of cucumber necrosis virus RNA ». Thesis, University of British Columbia, 1989. http://hdl.handle.net/2429/28969.
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The in vitro translation products directed by cucumber necrosis virus (CNV) RNA were analyzed in both rabbit reticulocyte lysate and wheat germ extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of ca. 33 Mr was produced. In wheat germ extracts, four proteins of ca. 41, 33, 21 and 20 Mr were produced. Hybrid-arrested translation (HART) studies using synthetic CNV antisense RNA corresponding to the entire CNV genome demonstrated that the four major proteins synthesized from CNV virion RNA in wheat germ extracts are virus-specific translation products. The genomic locations of the CNV in vitro translation products were determined using a number of experimental approaches including: (1) HART using antisense RNA corresponding to selected regions of the CNV genome; (2) in vitro translation of synthetic messenger-sense CNV transcripts; (3) immunoprecipitation of in vitro translation products with CNV polyclonal antisera and (4) in vitro translation of size-fractionated CNV virion RNA. Together, these experiments demonstrated that the ca. 33 Mr protein is derived from the 5' proximal coding region, the ca. 41 Mr protein is derived from an internal coding region, and that at least one but probably both of the ca. 20 and 21 Mr proteins are derived from the 3' terminal coding region(s) of the CNV genome. In addition, immunoprecipitation experiments provided further evidence that the ca. 41 Mr protein is the viral coat protein. The size, number, and genomic locations of the CNV in vitro translation products reported here are in agreement with those predicted from nucleotide sequence data (Rochon & Tremaine, 1989). The natural template for the expression of downstream cistrons in the CNV genome was investigated by in vitro translation of sucrose fractionated CNV virion RNA as well as in vitro translation of messenger-sense synthetic transcripts. These studies indicate that in vitro, both subgenomic and genomic-length CNV RNA molecules may act as templates for the synthesis of the ca. 41,21 and 20 Mr proteins as well as the ca. 33 Mr protein.Land and Food Systems, Faculty of
Graduate
9
Sun, Xiaojiao. « High Affinity Synthetic Molecular Binders for Proteins : Design, Synthesis and Evaluation ». Doctoral thesis, Uppsala universitet, Fysikalisk-organisk kemi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-183203.
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This thesis describes the design and synthesis of small molecule derivatives and their polypeptide conjugates as high affinity binders for proteins: the D-dimer protein (D-dimer), a biomarker for diagnosis of thromboembolic diseases; human myeloperoxidase (MPO), a biomarker for cardiovascular diseases; and chitinases, potential targets for asthma therapy. The interactions between the synthetic binder molecules and those proteins were evaluated by surface plasmon resonance (SPR) biosensor analysis and fluorescence spectroscopy. Competition SPR experiments or other methods proved that the small molecule components of the binder molecules were critical for binding and specifically bound to the original binding site of small molecules. The binder molecules consisted of a 42-residue helix-loop-helix polypeptide conjugated to a small molecule via aliphatic spacers of suitable length. The small molecules could be any type of moderately binding structure. In the binder development for the D-dimer, the tetrapeptide GPRP with a dissociation constant Kd of 25 μM was used and the affinity of 4C15L8GPRP obtained was estimated to be approximately 3 nM. In the binder development for MPO, salicylhydroxamic acid (SHA) with Kd of 2 μM was used and the affinity of 4C37L34C11SHA obtained was estimated to be approximately 0.4 nM. In the binder development for chitinases, a theobromine derivative (pentoxifylline) with a Kd of 43±10 μM was used and the affinity of 4C37L34-P obtained was estimated to be considerably higher than that of pentoxifylline. The binder molecules were identified from a 16-membered pool of candidates obtained by conjugating the small molecules to each member of a set of 16 designed polypeptides. The affinities were greatly enhanced by 2-3 orders of magnitude, compared to the small molecule. The polypeptides did not bind to the proteins with measurable affinities. The discovery of these new synthetic binders for protein targets can pave the way to diagnostic tests in vivo or in vitro, independent of antibodies.
10
Kim, Daniel. « Characterization of the Mata pre- ». Scholarly Commons, 2009. https://scholarlycommons.pacific.edu/uop_etds/738.
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Lindgren, Joel. « Chemical Engineering of Small Affinity Proteins ». Doctoral thesis, KTH, Proteinteknologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-141014.
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Small robust affinity proteins have shown great potential for use in therapy, in vivo diagnostics, and various biotechnological applications. However, the affinity proteins often need to be modified or functionalized to be successful in many of these applications. The use of chemical synthesis for the production of the proteins can allow for site-directed functionalization not achievable by recombinant routes, including incorporation of unnatural building blocks. This thesis focuses on chemical engineering of Affibody molecules and an albumin binding domain (ABD), which both are three-helix bundle proteins of 58 and 46 amino acids, respectively, possible to synthesize using solid phase peptide synthesis (SPPS). In the first project, an alternative synthetic route for Affibody molecules using a fragment condensation approach was investigated. This was achieved by using native chemical ligation (NCL) for the condensation reaction, yielding a native peptide bond at the site of ligation. The constant third helix of Affibody molecules enables a combinatorial approach for the preparation of a panel of different Affibody molecules, demonstrated by the synthesis of three different Affibody molecules using the same helix 3 (paper I). In the next two projects, an Affibody molecule targeting the amyloid-beta peptide, involved in Alzheimer’s disease, was engineered. Initially the N-terminus of the Affibody molecule was shortened resulting in a considerably higher synthetic yield and higher binding affinity to the target peptide (paper II). This improved variant of the Affibody molecule was then further engineered in the next project, where a fluorescently silent variant was developed and successfully used as a tool to lock the amyloid-beta peptide in a β-hairpin conformation during studies of copper binding using fluorescence spectroscopy (paper III). In the last two projects, synthetic variants of ABD, interesting for use as in vivo half-life extending partners to therapeutic proteins, were engineered. In the first project the possibility to covalently link a bioactive peptide, GLP-1, to the domain was investigated. This was achieved by site-specific thioether bridge-mediated cross-linking of the molecules via a polyethylene glycol (PEG)-based spacer. The conjugate showed retained high binding affinity to human serum albumin (HSA) and a biological activity comparable to a reference GLP-1 peptide (paper IV). In the last project, the possibility to increase the proteolytic stability of ABD through intramolecular cross-linking, to facilitate its use in e.g. oral drug delivery applications, was investigated. A tethered variant of ABD showed increased thermal stability and a considerably higher proteolytic stability towards pepsin, trypsin and chymotrypsin, three important proteases found in the gastrointestinal (GI) tract (paper V). Taken together, the work presented in this thesis illustrates the potential of using chemical synthesis approaches in protein engineering.QC 20140207
12
Brown, Angus R. « Solid phase synthesis of peptides and proteins ». Thesis, University of Edinburgh, 1995. http://hdl.handle.net/1842/27313.
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A strategy for the total chemical synthesis and purification of proteins has been investigated and applied to the 85 residue methylated DNA binding domain (MBD) from the chromosomal protein MeCP2, the 66 residue Restriction Alleviation (Ral) protein from bacteriophage λ and the 76 residue β-chemokine Monocyte Chemotactic protein (MCP-1). The hydrophobicity of the Nα protecting group tetrabenzo[a,c,g,i]fluorenyl-17-methoxycabonyl (Tbfmoc) has been exploited to simplify the rapid purification of the 85 amino acid MBD protein by Hplc. Initial structural studies on the synthetic protein are also reported. In addition a comparative study of semi-permanent, temporary and enzyme cleavable thiol protection has resulted in the extension of this Tbfmoc methodology to the synthesis of cysteine containing proteins such as Ral and MCP-1. A general route to C-terminal α-hydroxyglycine extended peptides via Fmoc/t-Bu based solid phase peptide synthesis is also described. Such peptides are the biosynthetic precursors of peptide amides in which the C-terminal carboxamide functionality is required for biological activity in a number of important hormones.
13
Wuu, Jessica Ja-li. « Cell-free synthesis of integral membrane proteins / ». May be available electronically:, 2007. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Yoo, Tae Hyeon Tirrell David A. Tirrell David A. « Proteins of novel composition : synthesis, evolution, dynamics / ». Diss., Pasadena, Calif. : California Institute of Technology, 2008. http://resolver.caltech.edu/CaltechETD:etd-03202008-163647.
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Neubauer, Cajetan Simon Johannes. « Structural aspects of quality control in protein synthesis ». Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609457.
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Misra, Rajeev. « Studies on the TolC protein of Escherichia coli K-12 and its effect on OmpF expression ». Title page, table of contents and abstract only, 1986. http://web4.library.adelaide.edu.au/theses/09PH/09phm678.pdf.
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Sadow, Jennifer Beth Hurley. « The X-ray crystal structure of wheat translation initiation factor eIF4E / ». Thesis, Full text (PDF) from UMI/Dissertation Abstracts International, 2002. http://wwwlib.umi.com/cr/utexas/fullcit?p3085056.
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Renberg, Björn. « Fluorescence-based ligand assays for protein detection using affibody affinity proteins ». Doctoral thesis, KTH, Skolan för bioteknologi (BIO), 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3936.
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The detection and quantification of biomolecules, and proteins in particular, are of great interest since these molecules are of fundamental importance to our well-being. Body fluids, as for instance human blood, are well suited for sampling of protein levels. However, the complexity of the fluids and the low abundance of many of the interesting biomolecules makes detection and quantification difficult. This has spurred an interest into the development of many protein detection methods, and of these, ligand assays have proven particularly suitable. In this thesis, different types of ligand assays for protein detection have been developed using affibody molecules as ligands. In a first study, a homogeneous competitive detection assay was investigated, based on antiidiotypic affibody molecule pairs and fluorescence resonance energy transfer (FRET) as reporting system. The individual members of two anti-idiotypic affibody pairs, each consisting of a target binding (idiotypic) and an anti-idiotypic affibody ligand, were labeled with a donor fluorophore and an acceptor fluorophore, respectively. Incubation with the two target proteins IgA and Taq DNA polymerase resulted in a concentration dependent decrease in the FRET signal, allowing for target protein detection and quantification. For Taq DNA polymerase, detection in 25% human plasma was also possible in the same concentration span as in buffer. In a second study, a homogeneous, non-competitive detection system was described. Affibody molecules of 58 amino acids directed against IgA and IgG were produced with chemical synthesis, and two fluorophores capable of FRET were site-specifically introduced. Binding of target protein induced a concentration-dependent change in the relative emission of the two fluorophores, which formed the basis for the detection system. In two studies, affibody molecules were evaluated and shown to function well as capture ligands on microarrays. Synthetic affibody molecules directed against Taq DNA polymerase and IgA were modified by the introduction of immobilization tags. Specific immobilization via a C-terminal cysteine or a biotin moiety, or random immobilization via amino groups, were studied in protein microarray experiments and SPR-based biosensor studies. The experiments showed that all immobilization chemistries resulted in functional capture molecules. A short spacer was also introduced, situated between the affibody and the cysteine and biotin moieties, which was shown to improve binding for all constructs. Multidomain affibody constructs of up to four N- to C-terminally linked domains were shown to increase the amount of bound target, compared to monomeric affibody ligands. Six dimeric affibody constructs directed against IgA, IgG, IgE, Taq DNA polymerase, TNF-α and insulin, respectively, showed low limits of detections for their targets and little or no cross-reactivity with the other target proteins. Dimeric affibody molecules directed against IgA and TNF-α were also shown to function in a sandwich format with antibodies for detection of targets in buffer and in human serum and plasma. Successful discrimination between normal and IgA-deficient sera showed that affibody molecules could be used for specific detection of protein in highly complex backgrounds on microarrays.QC 20100916
19
Haas, R. Matthew. « Synthesis and characterization of phosphono-CheY from Thermotoga maritima / ». Electronic version (PDF), 2007. http://dl.uncw.edu/etd/2007-1/haasr/rmatthewhaas.html.
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Cassidy, Peter Joseph. « The design and synthesis of peptide turn mimetics / ». St. Lucia, Qld, 1998. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16396.pdf.
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Tang, Chi-wai Sydney, et 鄧智偉. « Human kidney as an organ of complement synthesis : its regulation by tubular protein ». Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B31981768.
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Wei, Hen-Wei. « Effect of long-term amino-acid deficiency on the amino-acid composition of the body ». Thesis, University of Aberdeen, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302436.
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The aim of this study was to assess changes in the amino-acid composition of the body and in the fractional protein synthesis rates of individual organs or of haemoglobin as a result of long-term amino-acid deficiency in both growing and adult animals. The body weight loss of adults and the growth rates of growing animals were controlled by adjusting the degree of amino-acid deficiency according to individual body weight changes. Adult rats given amino acid-deficient diets had lower absolute weights of muscle (p = 0.052), liver (p = 0.002) and gastrointestinal tract (p < 0.001) than adequately-fed animals but chronic marginal amino-acid deficiency did not result in any measurable effect on whole-body amino-acid composition in comparison with well-nourished rats. In growing rats, liver weight, liver protein mass, fractional (% per day) and absolute (g / liver per day) synthesis rates of total liver protein, liver RNA activity and absolute albumin synthesis rate (g / liver per day) were depressed by long-term dietary deficiency of leucine, isoleucine or histidine. There were no significant differences in liver RNA capacities or relative albumin synthesis rates, however, between the adequate and the deficient groups. In growing pigs, deficiency of isoleucine, histidine or tryptophan depressed growth rate and the weights of liver, gastrointestinal tract, and muscle (biceps femoris). The fractional rates of total protein synthesis, both in individual tissues (muscle, skin and liver) and of haemoglobin, were also reduced. Pigs given histidine- or tryptophan- deficient diets had higher body hydroxyproline concentrations, suggesting changes in the deposition of collagen relative to other body proteins. The histidine-deficient pigs had lower whole-body histidine concentrations, due to absolute decreases in both haemoglobin and in free peptides rich in histidine. The histidine-deficient pigs also had low histidine concentrations in skin.
23
Narayanan, Pushpa. « Isolation and characterisation of novel ribosome-inactivating proteins from the root tubers of Trichosanthes kirilowii / Pushpa Narayanan ». HKBU Institutional Repository, 1996. http://repository.hkbu.edu.hk/etd_ra/67.
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Baldridge, Anthony Owen. « Synthesis, photophysics, and application of fluorescent protein chromophore analogs ». Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/44744.
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The green fluorescent protein chromophore exhibits remarkably different properties upon removal from the protective beta-barrel. This work focuses on the synthesis of these chromophores as wells studying the photophysics as to why they readily deactivate. Following these initial discoveries, these chromophores can be applied to many different environments providing a fluorescence "turn-on" and thus proving to be applicable in a number of different environments and fields.
25
Connors, Mark Terence. « The investigation of the parameters of protein synthesis and translational efficiency in the ovine / ». St. Lucia, Qld, 2002. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16926.pdf.
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Perera, Aruna B. Kane Robert R. « Modification of fresh tissue surfaces ; synthesis of labeled L-dopa analogs ; and synthesis of metoclopramide analogs / ». Waco, Tex. : Baylor University, 2005. http://hdl.handle.net/2104/2998.
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Nagalingam, Anil. « Towards the total synthesis of bacterial immunity proteins ». Thesis, University of Leeds, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.427784.
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Lear, Sam. « Total synthesis of bioactive peptides and whole proteins ». Thesis, Durham University, 2016. http://etheses.dur.ac.uk/11946/.
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Total chemical synthesis is an essential tool for the validation of natural product structures and the discovery and elaboration of novel therapeutic scaffolds. Chapter 1 (Part I) surveys existing treatments for trypanosomatid neglected tropical diseases, and the synthesis of a novel class of antiparasitic cyclic depsipeptides is reported (Chapter 2) alongside a full NMR assignment and structure calculation using NMR-derived distance restraints. The synthetic peptides exhibit activity profiles in agreement with published results, and a series of ester-to-amide substitution analogues also synthesized show similar low micromolar potency. Chapter 3 describes the synthesis of lassomycin, a tuberculocidal lasso peptide reported to exhibit a unique unthreaded topology. The naturally occurring peptide was synthesized alongside C-terminally amidated and truncated analogues, but none were biologically active. Given clear differences observed in the two-dimensional NMR data for synthetic lassomycin, it is suggested that the reported natural product in fact exists in the threaded form. The chemical synthesis of whole proteins is addressed in Part II, and current progress in the field is reviewed (Chapter 4). Work towards the total chemical synthesis of acyl carrier protein using a two fragment approach is described in Chapter 5. The N-terminal fragment was synthesized using the sulfonamide linker, and while the C-terminal fragment presented difficulties due to extremely low solubility, solubilization using backbone protection was demonstrated. The development of a modified Dawson linker for the synthesis of peptide N-acylureas without overacylation is also described. Finally, Part III details the synthesis of tumor targeting peptides used for imaging and inhibition of cancer cell growth, and which cause tumor size reduction in vivo. A novel web utility used for the automated assignment of peptide mass spectra throughout this thesis is also presented.
29
Mitchell, Christopher J. « The regulation of polyphosphoinositide synthesis in rat liver ». Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389879.
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Zhang, Guangtao. « Design, synthesis, and evaluation of cholera toxin inhibitors and [alpha]-helix mimetics of dormancy survival regulator / ». Thesis, Connect to this title online ; UW restricted, 2006. http://hdl.handle.net/1773/8485.
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Thompson, Robert E. A. « Strategies for the synthesis of posttranslationally modified peptides and proteins ». Thesis, The University of Sydney, 2014. http://hdl.handle.net/2123/11435.
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Posttranslational modifications (PTMs) are ubiquitous chemical alterations that are made to peptides and proteins after ribosomal synthesis. The non-templated nature of PTMs hinders the ability to study an individual isoform of a given protein, which may be expressed as complex mixtures in vivo. This thesis describes ligation-based strategies for use in the chemical synthesis of PTM peptides and proteins. Chapter one describes the development of a synthesis of the antibacterial PTM peptide microcin B17 (MccB17). Using Ag(I)-ligation chemistryMccB17 was synthesised efficiently from three precursors. This strategy permitted the synthesis of 7 analogues for use in structure-activity relationship studies. A truncated analogue of MccB17 was shown to be a more potent poison of DNA gyrase, the target of MccB17, compared with MccB17. Chapter two describes ligation–desulfurisation chemistry at β-SH aspartate (Asp). A protected form of β SH Asp was accessed efficiently in 3-steps for direct use in peptide synthesis. Ligation reactions of peptides bearing this amino acid were rapid and ligated products could be desulfurised to afford peptides bearing the native Asp residue in high yields. Computational analysis then prompted exploration of chemoselective desulfurisation of β-SH Asp in the presence of Cys residues. This strategy permitted the synthesis of the N-terminal extracellular domain of CXCR4, bearing a sTyr residue and an N-linked glycan. Chapter three outlines the use of a new alkyl thiol additive for use in one pot ligation–desulfurisation chemistry, namely 2,2,2-trifluoroethanethiol (TFET). This alkyl thiol facilitated ligation rates similar to the current gold standard thiol additives. Importantly, the alkyl nature of TFET allowed desulfurisation to be conducted in the same vessel, a feat not possible with aryl thiol additives. Through the use of TFET, the synthesis of four sulfoforms of the 60 amino acid (sulfo)protein madanin-1 was achieved.
32
Ghannad, Mona. « Design and Synthesis of Collagen-binding Anti-microbial Proteins ». Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/19981.
Texte intégralRésumé :
The Herpes simplex virus (HSV) is a virus that commonly infects the skin, and mucous membrane of the mouth, genitalia, and the eye. HSV-1 is the strain that is most commonly associated with corneal infections, and it is the most frequent cause of corneal blindness in North America [1]. Currently no cure is available, and many limitations are characterized by the currently available synthetic antiviral drugs, which suggest the need for other potential drug alternatives and delivery strategies. Anti-microbial peptides are naturally occurring peptides that are potent killers of a broad range of micro-organisms, including bacteria, fungi, and viruses [2]. AMPs are known to be a key component of the innate immune response at the human ocular surface. The human cathelicidin-derived AMP, LL-37, expressed in human corneal epithelial cells provides a wide range of protection against viral pathogens such as HSV-1 [3]. My thesis research addressed the design and recombinant production of hybrid AMP sequences containing LL-37 with the potential ability to form chemical or physical associations with a Collagen scaffold material, such as those used in current artificial cornea constructs to address the need for alternative anti-viral drugs. Three fusion proteins were tested, and compared for feasible design anti-microbial peptide expression and purification in E. coli. It was illustrated that the thioredoxin and SUMO fusion systems are good candidates for successful recombinant production of active designed peptides. The point-mutated LL-37 sequence was successfully expressed and purified using the thioredoxin fusion system. It was demonstrated that this modified LL-37 was effective against HSV-1 infection. The SUMO system was used to express the bio-functional LL-37 containing a collagen-binding sequence. Further work is required to address issues regarding recombinant AMP production, such as increasing enzymatic cleavage efficacy, and minimizing proteolytic degradation or modification.
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Kotaras, Peter J. « Synthesis and secretion of rat pineal proteins in-vitro / ». Title page, contents and abstract only, 1992. http://web4.library.adelaide.edu.au/theses/09PH/09phk871.pdf.
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Hui, Daniel Jason. « The Mechanism of Protein Synthesis Inhibition by the P56 Family of Viral Stress Inducible Proteins ». Connect to text online, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1104848977.
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Dingwall, C. « The accumulation of proteins in the Xenopus oocyte nucleus ». Thesis, Open University, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.354967.
Texte intégralRésumé :
The ability of proteins to accumulate in the nucleus has been studied by injecting nucleoplasmin and calf thymus histone H1 into the cytoplasm of Xenopus oocytes. Nucleoplasmin, the most abundant protein in the Xenopus oocyte nucleus is pentameric and proteolysis of the nuceloplasmin pentamer produces a relatively protease resistant 'core' molecule that cannot enter the nucleus after microinjection into the cytoplasm. The polypeptide domain ('lq tail') of each subunit removed by proteolysis was obtained as a discrete fragment and has the ability to accumulate in the nucleus. Partially cleaved pentameric molecules with a single intact sub unit can still accumulate in the nucleus. Therefore a polypeptide domain of nucleoplasmin has been found that is both necessary and sufficient for accumulation in the nucleus. When the `core' molecule was injected directly into the oocyte nucleus it remained there, indicating that the 'tail' region confers selective entry rather than selective retention. In the case of histone H1 a proteolytic fragment encompassing the carboxyterminal domain can accumulate in the nucleus. The amino acids lysine, proline and alanine comprise 75 of the 89 amino acids in this fragment. Since the remaining 14 amino acids are scattered throughout the fragment and not clustered any primary sequence specifying entry into the nucleus would seem necessarily to involve the amino acids lysine, proline and alanine. Positive charge alone cannot explain the accumulation of this gragment since poly L-lysine does not accumulate after microinjection into the cytoplasm. Fragments encompassing other domains of the molecule are so unstable in the oocyte that their ability to accumulate in the oocyte nucleus cannot be assayed. The gene for nucleoplasmin has been cloned and sequences have been found in the 'tail' region of nuceloplasmin that show homology to sequences identified in other nuclear proteins that appear to constitute a signal specifying nuclear localisation.
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Huang, Edwin P. C. Biotechnology & Biomolecular Sciences Faculty of Science UNSW. « Recombinant protein production utilising a metallothionein expression system and a Super-CHO cell line ». Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences, 2006. http://handle.unsw.edu.au/1959.4/24940.
Texte intégralRésumé :
A novel metal-inducible and amplifiable metallothionein (MT) expression system, pNK, was firstly optimised and characterised for the production of a reporter protein, human growth hormone (hGH) in a suspension CHO cell line grown in a serum-free media. The pNK-based hGH production was demonstrated in cadmium-free condition under various fermentation modes (batch, fed-batch and perfusion) and scales (flask to bench-top bioreactor). Improvement of specific productivity of recombinant protein from pNK was shown to be possible by addition of butyrate or substrate substitution of glutamine by glutamate. Combination of fed-batch and butyrate addition strategies resulted in more than one gram per litre of hGH being obtained from the pNK expression system in a bioreactor. In the second part of the project, based on a statistical approach suggested by Plackett-Burman (P-B), a chemically-defined and protein-free medium, named Super-CHO protein-free (SPF), was developed to support a Super-CHO cell line, C2.8-325, to grow as a single-cell suspension culture with comparable growth rate and viable cell number as observed in a commercial medium containing undefined additives. Using Dulbecco's Modified Eagle's Medium/Ham's F12 1:1 mixture (DMEM/F12) as the basal medium, a P-B design matrix screened 10 nutritional components. Components shown potentially beneficial for cell growth rate and viable cell number were supplemented to DMEM/F12 to formulate the SPF medium. Finally, the pNK expression system and the Super-CHO cell line were applied simultaneously in an attempt to express a humanised anti-CD48 monoclonal antibody (MAb), IgG1-N2A (N2A-MAb). This aimed to test C2.8-SPF grown in newly developed SPF medium for transfection, clone development and recombinant protein production. A stable and N2A-MAb expressing C2.8-SPF cell line was successfully constructed, and N2A MAb expression was subsequently amplified and demonstrated in various cultivation scales (flask and bioreactor). This project demonstrated that the novel metal-inducible and - amplifiable mammalian expression system, pNK, and the novel mammalian host cell-line, Super-CHO C2.8-SPF, capable of growing as a single-cell suspension culture in a chemically-defined protein-free medium, SPF, could be utilised in combination to provide a new, low-cost, and regulatory-compliant recombinant protein expression platform, suitable for the biopharmaceutical industry to use in the manufacture of therapeutic recombinant proteins.
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Adedeji, Dolapo A. Duin Evert C. « Isoprenoid synthesis new roles for iron sulfur clusters / ». Auburn, Ala., 2007. http://repo.lib.auburn.edu/Send%2002-04-08/ADEDEJI_DOLAPO_4.pdf.
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Tang, Norina Mei Ngon. « Regulation of protein synthesis and induction of oncogenesis by a cellular protein kinase inhibitor / ». Thesis, Connect to this title online ; UW restricted, 1998. http://hdl.handle.net/1773/11501.
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Tang, Chi-wai Sydney. « Human kidney as an organ of complement synthesis : its regulation by tubular protein / ». Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk:8888/cgi-bin/hkuto%5Ftoc%5Fpdf?B23295181.
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Suleman, Essa. « Mutational analysis of the PacC binding sites within the aflR promoter in Aspergillus flavus ». Thesis, Nelson Mandela Metropolitan University, 2011. http://hdl.handle.net/10948/d1012683.
Texte intégralRésumé :
It is generally known that media containing simple sugars (sucrose, glucose) and organic nitrogen sources (ammonium) when buffered to acidic pH stimulates aflatoxin production in Aspergillus flavus & A. parasiticus while lactose, nitrate and an alkaline pH inhibit aflatoxin biosynthesis. It has been shown that pH of the growth medium is the most important regulatory factor for aflatoxin biosynthesis since media containing stimulatory carbon and/or nitrogen sources (sucrose and ammonia) do not enhance aflatoxin (or sterigmatocystin) production at alkaline pH. RNA interference (in A. flavus) of the pH regulatory transcription factor, PacC, resulted in aflatoxin production under acidic and alkaline pH conditions whilst wildtype Aspergillus flavus produced aflatoxins only under acidic conditions. This conclusively proved that PacC negatively regulates aflatoxin production at alkaline pH in A. flavus. However the exact mechanism involved in PacC repression of aflatoxin biosynthesis at alkaline pH still remains unknown. The AflR protein is essential for expression of several genes in the aflatoxin biosynthetic cluster. In the current study, sequence analysis of the aflR promoter indicated the presence of two putative PacC binding sites within the aflR promoter of A. flavus 3357WT located at positions -162 and -487 bp from the start codon. The presence of the PacC binding sites in the aflR promoter indicated a possible link between aflR expression and PacC regulation under alkaline conditions. Thus, in this study, it was hypothesized that at alkaline pH, PacC inhibits aflR expression by binding to one or both of the PacC binding sites within the aflR promoter. This in turn, would result in inhibition of aflatoxin biosynthesis since expression of several aflatoxin biosynthetic pathway genes is dependent on activation by AflR. The aim and objective of this study was to test the validity of this hypothesis i.e. that at alkaline pH PacC binds to one or both of its recognition sites within the aflR promoter thereby inhibiting aflR expression which subsequently would result in inhibition of aflatoxin biosynthesis. This was done by first mutating each individual and then both PacC binding sites in the A. flavus 3357 aflR promoter via Single-Joint PCR (SJ-PCR) and fusing the wildtype and each mutated aflR promoter to the Green Fluorescent Protein (gfp) gene and the trpC terminator to yield a functional expression vector. These constructs were then transformed into A. flavus 3357.5. Positive transformants were confirmed to express GFP by fluorescence microscopy and spectrofluorometry. Quantification of GFP protein levels of the various transformants in this study indicated that PacC negatively regulated aflR promoter activity at alkaline pH. RT-qPCR was performed on positive transformants after growth on SLS medium at acidic and alkaline pH to determine if PacC negatively regulated aflR promoter activity at alkaline pH and to determine whether PacC binds preferentially to one or both recognition sites within the aflR promoter. RT-qPCR analysis suggest that PacC binds non-preferentially to both recognition sites within the aflR promoter on sucrose and lactose media at alkaline pH, although mutation of PacC binding site 2 results in a slightly higher expression compared to mutation of PacC binding site 1. Increasing the concentration of an aflatoxin conducive nitrogen source stimulated aflR promoter activity but this was not sufficient to overcome negative regulation by PacC. It is generally known that repression of aflR expression results in repression of aflatoxin biosynthesis irrespective of pH. The results of this study strongly suggest that PacC negatively regulates aflR promoter activity at alkaline pH by binding to one or both PacC recognition sites within the aflR promoter. Since aflR promoter activity is repressed by PacC at alkaline pH, this substantiates the hypothesis that PacC represses aflatoxin biosynthesis by inhibiting expression of aflR. Furthermore, the results of this study indicated that there may be some PacC protein present in the active form at acidic pH irrespective of the carbon source and nitrogen source used in the growth medium. RT-qPCR analysis indicated that any active PacC present at acidic pH may cause repression of the aflR promoter based on the position of the PacC binding site relative to the aflR start codon, although it appears that PacC may have a higher affinity for PacC binding site 2 (which is closer to the aflR start codon).
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Schinn, Song Min. « Cell-Free Synthesis of Proteins with Unnatural Amino Acids : Exploring Fitness Landscapes, Engineering Membrane Proteins and Expanding the Genetic Code ». BYU ScholarsArchive, 2017. https://scholarsarchive.byu.edu/etd/6496.
Texte intégralRésumé :
Unnatural amino acids (uAA) expand the structural and functional possibilities of proteins. Numerous previous studies have demonstrated uAA as a powerful tool for protein engineering, but challenges also remain. Three notable such challenges include: (1) the fitness of uAA-incorporated proteins are difficult to predict and time-consuming to screen with conventional methods, (2) uAA incorporation in difficult-to-express proteins (e.g. membrane proteins such as G-protein coupled receptors) remain challenging, and (3) the incorporation of multiple types of uAA are still limited. In response, we pose cell-free protein synthesis (CFPS), a rapid and versatile in vitro expression system, as a platform to explore solutions to these challenges. The "cell-free" nature of CFPS enables it to accelerate protein expression and tolerate extensive modifications to its translational environment. In this work, these advantages were utilized to address the aforementioned challenges by: (1) rapidly expressing and screening uAA-containing proteins, (2) incorporating uAA in functional G-protein coupled receptor in the presence of membrane-mimicking lipid additives, and (3) engineer the translational environment extensively towards multiple uAA incorporation.
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Vinogradov, Alexander Alexandrovich. « New methods for synthesis and modification of peptides and proteins ». Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/109680.
Texte intégralRésumé :
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2017.Cataloged from PDF version of thesis.
Includes bibliographical references.
Chemical modification of peptides and proteins is an enabling suite of tools for tailoring the properties of these biomolecules to specific applications. A number of bio-conjugation reactions allows fine-tuning of the biological activity, proteolytic stability, and immunogenicity of peptides and proteins, as well as equipping them with completely novel functions such as cell penetration, fluorescence, unique chemical reactivity, and much more. Described herein are a number of new methods for the synthesis of modified peptides and proteins, and an approach to the discovery of such methodologies. Applications of fast-flow solid phase peptide synthesis - a technique recently developed to accelerate and improve peptide synthesis- towards the synthesis of difficult sequences and the refinement of associated protocols is described. The utility of the system is demonstrated via rapid total synthesis of barnase, a model 110-residue RNase, in the L- and D-forms. Systematic characterization of the biochemical properties of the synthesized proteins revealed that barnase is able to hydrolyze substrates of various chiralities, and that D-barnase is fully proteolytically stable. Separately, a method for the preparation and utilization of unprotected peptide isocyanates in water was developed. It was shown that easily accessible C-terminal peptide isocyanates can be conjugated to a number of strong nucleophiles in the presence of unprotected amino acid side chains for peptides and proteins of various structures. Two-component macrocyclization of peptide isocyanates with bifunctional linkers was developed as an extension of the described chemistry. The resulting cyclic peptides were shown to be more proteolytically stable and more bioactive than their linear analogs. In pursuit of generalizing the C-terminal protein modification chemistry to fully proteogenic peptides and proteins, a number of library screening approaches was developed. Liquid chromatography coupled to tandem mass spectrometry was employed to screen and reliably decode synthetic peptide libraries in a high-throughput manner. These protocols were used to discover proteogenic sequence tags reactive towards substituted hydrazine derivatives in a transpeptidation reaction. The discovered C-terminal tripeptide tag His-Gly-Cys underwent transpeptidation with a number of structurally different nucleophiles in various sequence contexts.
by Alexander Alexandrovich Vinogradov.
Ph. D.
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Patterson, Jennifer Ann. « The chemical synthesis of proteins and peptide C-terminal derivatives ». Thesis, University of Edinburgh, 1999. http://hdl.handle.net/1842/12777.
Texte intégralRésumé :
Methodology for the synthesis of peptide C-terminal aldehydes has been investigated. Modification of a linker system, based on a terabenzosuberyl construct, has been demonstrated to be suitable for the synthesis of peptide C-terminal semicarbazones. The route has been fully optimised to yield a series of peptide C-terminal semicarbazones and the corresponding peptide aldehydes. The chemical synthesis of deglycosylated human interferon-gamma (143 residues) has been carried out. The purification of this protein has been investigated and a short purification protocol developed which is sufficiently general to allow application to other similar protein systems. Purification and characterisation of the synthetic interferon-gamma molecule has been completed and folding of the molecule attempted.
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Hsieh, Yves Shang-Yi. « Chemical Synthesis of Peptides & ; Proteins with Post-Translational Modifications ». Thesis, The University of Sydney, 2013. http://hdl.handle.net/2123/9422.
Texte intégralRésumé :
This thesis describes efforts aimed at the synthesis of peptides and proteins bearing homogeneous carbohydrate and sulfate modifications for interrogation of the structural and functional roles of these post-translational modifications. This thesis is divided into two parts. The first section (Chapter Two and Chapter Three) describes efforts toward the synthesis of glycopeptides. Chapter Two describes the synthesis of rare S-linked glycopeptide sublancin-168 and its glycosylated variants, along with structural studies to understand the effect of S-glycosylation. Chapter Three outlines the isolation of N-linked oligosaccharides from a natural source in large quantity, and the study towards the utility of these isolated N-linked glycans to the synthesis of N-linked glycopeptides using a solid-phase aspartylation approach. The second section (Chapter Five and Chapter Six) of this thesis is devoted to the total synthesis of hirudin P6 peptide fragments and proteins bearing homogeneous carbohydrate and sulfate modifications. Inhibitory assays of these peptides and proteins against thrombin confirmed the importance of tyrosine sulfation for fibrinogenolytic activity via binding to exosite I of the protein. Introduction of both sulfation and/or O-glycosylation was shown to have a detrimental effect on binding and inhibition of the amidolytic activity of thrombin.
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Blanshard, Maximilian Edward Alfred. « Synthesis of recombinant antibacterial proteins in the Chlamydomonas reinhardtii chloroplast ». Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10045326/.
Texte intégralRésumé :
The rise of antibiotic resistance and the decline in antibiotic discovery have been well publicised. These issues, in combination with a growing global reliance upon antibiotics for everyday modern life, urgently require the discovery of novel antibacterial drugs. Endolysins are one potential candidate to support, or replace, conventional antibiotics. Endolysins are lytic enzymes produced by bacteriophage in natura to enable the release of viral progeny from inside the host bacterium. When applied exogenously, endolysins can lyse Gram-positive bacteria, and thus could be used as a novel antibacterial for this group of pathogens. Biologically active endolysins have been successfully expressed as recombinants in the chloroplast of the green alga, Chlamydomonas reinhardtii. C. reinhardtii, and in particular the chloroplast, has several features as a cell factory which make it an attractive alternative to the traditional recombinant protein production platforms. C. reinhardtii is free of endotoxins, can be cultivated at low cost in photobioreactors, has GRAS status, and is genetically tractable. This study initially focuses upon improving the accumulation and activity of one endolysin, Cpl-1, targeting Streptococcus pneumoniae. Recombinant Cpl-1 has been shown previously in the Purton lab to accumulate to moderate levels in the C. reinhardtii chloroplast. Here we present two transgenic lines of C. reinhardtii that appear to accumulate recombinant Cpl-1 to higher levels – one through the incorporation of multiple expression cassettes, and one through codon pair optimization. To improve the activity of Cpl-1 as an enzyme, Cpl-1 binding site mutagenesis, Cpl-1 dimerization, and the production of a potentially synergistic holin protein were all attempted. Finally, an endolysin against Clostridium difficile, CD27L, was successfully produced in the C. reinhardtii chloroplast and shown to be active in vitro. Another endolysin, this time targeting Propionibacterium acnes, failed to express in C. reinhardtii, but was expressed in E. coli, albeit without obvious lytic activity.
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Patel, Amar S. « Synthesis of Aromatic Monothiols and Aromatic Dithiols to Increase the Folding Rate and Yield of Disulfide Containing Proteins ». FIU Digital Commons, 2010. http://digitalcommons.fiu.edu/etd/313.
Texte intégralRésumé :
Most pharmaceutically relevant proteins and many extracellular proteins contain disulfide bonds. Formation of the correct disulfide bonds is essential for stability in almost all cases. Disulfide containing proteins can be rapidly and inexpensively overexpressed in bacteria. However, the overexpressed proteins usually form aggregates inside the bacteria, called inclusion bodies, which contains inactive and non-native protein. To obtain native protein, inclusion bodies need to be isolated and resolubilized, and then the resulting protein refolded in vitro. In vitro protein folding is aided by the addition of a redox buffer, which is composed of a small molecule disulfide and/or a small molecule thiol. The most commonly used redox buffer contains reduced and oxidized glutathione. Recently, aliphatic dithiols and aromatic monothiols have been employed as redox buffers. Aliphatic dithiols improved the yield of native protein as compared to the aliphatic thiol, glutathione. Dithiols mimic the in vivo protein folding catalyst, protein disulfide isomerase, which has two thiols per active site. Furthermore, aromatic monothiols increased the folding rate and yield of lysozyme and RNase A relative to glutathione. By combining the beneficial properties of aliphatic dithiols and aromatic monothiols, aromatic dithiols were designed and were expected to increase in vitro protein folding rates and yields. Aromatic monothiols (1-4) and their corresponding disulfides (5-8), two series of ortho- and para-substituted ethylene glycol dithiols (9-15), and a series of aromatic quaternary ammonium salt dithiols (16-17) were synthesized on a multigram scale. Monothiols and disulfides (1-8) were utilized to fold lysozyme and bovine pancreatic trypsin inhibitor. Dithiols (11-17) were tested for their ability to fold lysozyme. At pH 7.0 and pH 8.0, and high protein concentration (1 mg/mL), aromatic dithiols (16, 17) and a monothiol (3) significantly enhanced the in vitro folding rate and yield of lysozyme relative to the aliphatic thiol, glutathione. Additionally, aromatic dithiols (16, 17) significantly enhance the folding yield as compared to the corresponding aromatic monothiol (3). Thus, the folding rate and yield enhancements achieved in in vitro protein folding at high protein concentration will decrease the volume of renaturation solution required for large scale processes and consequently reduce processing time and cost.
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Ellsmore, Victoria. « Human cytomegalovirus origin-dependent DNA synthesis ». Thesis, University of Glasgow, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340332.
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Dodd, Sara. « Hydrodynamic and hydrogel properties of mucins from cultured guinea-pig tracheal epithelial cells ». Thesis, University of Nottingham, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.287166.
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Gillespie, Charles Stewart. « Myelin membrane protein biosynthesis : an in vitro study ». Thesis, University of Stirling, 1988. http://hdl.handle.net/1893/22868.
Texte intégralRésumé :
The sites of biosynthesis and incorporation of the abundant CNS myelin proteins 2' , 3' -cyclic nucleotide-3'-phosphodiesterase (CNPase) and P2 protein into the growing myelin membrane were investigated. Cell-free translation systems programmed with mRNA from rat brain, rabbit spinal cord, free and bound polysomes and purified myelin demonstrated conclusively that both CNPase and P2 are synthesized on free polysomes like the myelin basic proteins (MBPs) but unlike the proteolipid protein (PLP), the major intrinsic membrane protein of CNS myelin, which is known to be synthesized at the oligodendrocyte endoplasmic reticulum on bound polysomes (Colman et al., 1982) . These observations were supported by labelling studies on rats in vivo during the period of maximal myelin deposition. Newly synthesized CNPase associated with the myelin membrane very rapidly after labelling (~2 minutes) and this is consistent with the view that there is only a brief delay between synthesis and incorporation into their target membrane for extrinsic-type plasma membrane proteins. An RNA fraction isolated from purified CNS myelin was not enriched in mRNAs coding for CNPase and P2 but a considerable enrichment of mRNAs coding for MBPs was observed. This phenomenon has important implications for the cell biology of myelination since it suggests that although MBPs, CNPase and P2 are all basic extrinsic membrane proteins, and synthesized on free polysomes, different mechanisms for their transport to the myelin membrane exist. The addition of dog pancreatic microsomes (DPM) during translation showed no membrane association for CNPase however, at least 50% of MBPs were observed to non-specifically associate with these membranes. When newly synthesized MBP and P2 were incubated post-translationally with DPM or rabbit spinal cord myelin P2 only associated with myelin whereas MBP showed an equal affinity for both types of membranes. The segregation of MBP free polysomes at the myelin membrane during synthesis ensures that the nascent MBP polypeptides associate with the correct membrane. Recent evidence has shown that the free polysome-mRNA complex is bound to the cytoskeleton during protein synthesis. After extensive characterization of the purified rat brain oligodendrocyte and myelin-associated cytoskeletons it was shown that the synthesis of MBPs and CNPase only occurs from mRNA that is associated with the cytoskeleton and not when it is part of the cytoplasmic mRNA pool. Lipid analysis of the purified rat brain myelin-associated cytoskeleton revealed the presence of tightly bound lipid with a considerable enrichment of cerebroside and sphingomyelin (the latter at the expense of phosphatidylethanolamine). These studies on the cytoskeletal involvement in myelinogenesis suggest that extrinsic CNS myelin proteins are synthesized on the cytoskeleton and that post-translational cytoskeletal transport of these proteins to the growing myelin membrane may take place.
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Pfeffer, Frederick Matthew, et mikewood@deakin edu au. « Approaches to the synthesis of peptide substituted frameworks ». Deakin University. School of Biological and Chemical Sciences, 2000. http://tux.lib.deakin.edu.au./adt-VDU/public/adt-VDU20060713.125622.
Texte intégralRésumé :
The 1,3 dipolar cycloaddition between carbonyl ylids (generated from cyclobutene epoxides flanked by esters) and norbornyl alkenes the ACE reaction offers a facile method for the construction of polynorbornyl molecular frameworks. This reaction has, as described in this dissertation, underpinned the construction of molecular frameworks that have peptides and amino acids attached. Such highly rigid peptide-frameworks are of use in the field of peptidomimetics; the template molecule governs the final positioning of any attached groups such that a precise arrangement of amino acids can be achieved without the need to construct entire proteins.
In the course of any ACE reaction the ester flanked cyclobutene epoxide is transformed to a 1,3 dipole, the esters serve to stablise this reactive intermediate and are as a consequence incorporated in the reaction product. Modification of these esters provides pseudo-equatorial points for peptide attachment. These methyl esters were replaced with tert-butyl esters to provide pseudo-axial attachment points that could be selectively addressed.
The optimal strategy for peptide-framework construction involved direct condensation of carboxyl protected amino acids to bicyclo[2.2.1]hept-5-ene-2-endo-carboxylic acid as well as condensation of amino acids to cyclobutene epoxides derived from this acid. The ACE reaction of (±) bicycloheptene-2-endo-carboxylic acid derivatives with cyclobutene epoxides synthesised from such racemic acid derivatives provided a mixture of enantiomers and meso compounds. In order to control the position of the attachment points and hence the final location of the attached peptides the ACE reaction required chiral starting materials. Accordingly, all peptidoframeworks were derived from the chiral (2S)-(-)-bicycloheptene carboxylic acid. The ACE reaction of this (S)-norbornene with the (S)-epoxide provided a peptide framework in which the attached amino acids were positioned pseudo-axially. Deprotection of the amino acid allowed peptide chain building in the pseudo-axial direction. Using this strategy a framework with an alanine residue and a triglycine peptide was synthesised. By combining this strategy with the ter-butyl ester variant a framework with pseudo-axial alanine and pseudo-equatorial glycine residues was manufactured.