Lipase B from Pseudozyma antarctica (PalB), had been expressed in several recombinant protein hosts and showed very good transesterification activity for biodiesel production. However, the functional expression could not be demonstrated until recently in the most popular recombinant protein expression system, e.g. Escherichia coli, and the expression performance stands improvement. The probable reason is that PalB is a lipase with more hydrophobic surface and three disulfide bonds and thus may not be easily expressed in E. coli functionally. This thesis focuses on the identification of factors limiting heterologous expression of PalB in E. coli through a systematic study by using several strategies, including the different expression compartments, fusion tags, folding factors, and host strains. Functional expression of PalB in the cytoplasm of E. coli was explored using BL21(DE3) and its mutant derivative Origami B(DE3) as the host. Bioactive PalB was obtained in the reduced cytoplasm of BL21(DE3), implying that the formation of disulfide bond was not strictly required for functional expression. However, the expression was ineffective and was primarily limited by formation of PalB inclusion bodies and growth arrest, both of which were associated with PalB misfolding and deteriorated physiology. The culture performance in terms of cell growth and PalB expression level could be significantly improved by simultaneous coexpression of multiple chaperones of trigger factor and GroEL/ES, but not individual coexpression of either one of them. It was proposed that the two chaperones mediate the early stage and late stage of cytoplasmic PalB folding and would be required simultaneously for boosting both the overall PalB synthesis rate and the cytoplasmic folding efficiency. On the other hand, a much higher bioactive PalB was produced in Origami B(DE3) harboring the same PalB expression vector. Furthermore, the significant high bioactive PalB was produced by coexpression of periplasmic folding factor without a signal peptide (e.g., coexpression of DsbA, and DsbC). Coexpression of DsbA was found to be effective in enhancing PalB expression and such an improvement was more pronounced in Origami B(DE3), suggesting that both folding and disulfide bond formation could be the major factors limiting PalB expression. The fusion tag technique was also explored by constructing several PalB fusions for the evaluation of their expression performance. While the solubility was enhanced for most PalB fusions, only the DsbA tag was effective in boosting PalB activity possibly via both enhanced solubility and correct disulfide bond formation. Our results suggest that solubilization of PalB fusions did not necessarily result in the development of PalB activity which could be closely associated with correct disulfide bond formation. While PalB was stably expressed in the cytoplasm, most of the expressed gene product aggregated in cells as inactive inclusion bodies. In contrast, PalB was extremely unstable when expressed in the periplasm, also leading to poor expression performance. Such unstable PalB can be rescued by coexpression of several periplasmic folding factors, such as DegP, FkpA, DsbA, and DsbC, but not cytoplasmic chaperones. As a result, the performance for functional PalB expression in the periplasm was significantly improved. This is the first report demonstrating the use of folding factors to rescue the extremely unstable gene product that is otherwise completely degradable. On the other hand, functional expression of PalB in the periplasm was explored using four fusion tags, e.g., DsbC, DsbA, maltose binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags for functional expression of PalB, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of gene products, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegPS210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance for functional expression of PalB was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/liter. Without extracting protein from cells the whole cell can be directly used as a platform for the immobilized enzyme. Proof-of-concept experimentation was conducted by PalB display on the E. coli cell surface. By fusing the palB gene in between the signal peptide phoA and an autotransporter Protein EstA’s gene under the lac promoter, PalB was successfully displayed on the E. coli cell surface. However, cells encountered a severe physiological stress. Coexpression of various periplasmic folding factors, e.g., DegP, SurA, DsbA and DsbC could erease the physiological stress, but only DsbA was demonstrated to be effective to restore cell physiology and increase PalB expression level. Key words: enzyme, Escherichia coli, chaperone, folding factor, fusion tag, gene expression, Pseudozyma antarctica, lipase B, recombinant protein production

Rhesus macaques represent a valuable model in biomedical research and in development of vaccines and therapeutics. Due to the lack of reagents, the general properties of IgG and corresponding cellular receptors (FcγR) in this species are poorly characterized. We engineered recombinant IgGs containing each of the four rhesus macaque heavy constant region (CH) subclasses. To define FcγRs that mediate IgGs, we identified and characterized three FcγR classes, and generated recombinant cDNA constructs. cDNA IgH constructs were created by fusing – by sequence overlap extension PCRs – a gene segment encoding the murine variable heavy domain specific for the hapten NIP, an established specificity system for assessing antibody effector functions, with rhesus macaque CH fragments. The complete IgH constructs were transfected into J558L cells, a murine IgH-lost myeloma cell line expressing anti-NIP light chain. Secretion of engineered IgGs was determined by ELISAs using NIP-BSA and anti-monkey IgG-specific antibodies. Molecular cloning methods were applied to identify and clone FcγR genes, and recombinant FcγR cDNA constructs were created by the recombinant DNA method. Four engineered IgH cDNA constructs were successfully created. Recombinant IgGs, in the intact Ig form and retaining the original anti-NIP specificity, were successfully produced. Compared to those in humans, FcγRs in rhesus macaques share high homology, yet also feature a relatively high level of intra-species polymorphism and possess different N-linked glycosylation patterns. FcγR constructs and expression vectors were successfully generated. The chimeric recombinant IgGs are powerful tools for defining IgG functional properties and studying CH structure/function relationship. These molecules can also be used as immunogens for generation of antibodies capable of unequivocally detecting individual IgG subclasses. The findings on FcγRs validate rhesus macaques as a model for studying antibody responses, and underscore the need to take into account of the genetic heterogeneity. The FcγR constructs and vectors serve as a tool for further studies of IgG/FcγR interactions. We also reported here our findings from a separate study that the main female hormone, 17β-estradiol, is capable of restoring antibody responses to an influenza vaccine in a postmenopausal mouse model, suggesting that immunogenicity and efficacy of influenza vaccines should be evaluated in postmenopausal women.

Lipase B from Pseudozyma antarctica (PalB), had been expressed in several recombinant protein hosts and showed very good transesterification activity for biodiesel production. However, the functional expression could not be demonstrated until recently in the most popular recombinant protein expression system, e.g. Escherichia coli, and the expression performance stands improvement. The probable reason is that PalB is a lipase with more hydrophobic surface and three disulfide bonds and thus may not be easily expressed in E. coli functionally. This thesis focuses on the identification of factors limiting heterologous expression of PalB in E. coli through a systematic study by using several strategies, including the different expression compartments, fusion tags, folding factors, and host strains. Functional expression of PalB in the cytoplasm of E. coli was explored using BL21(DE3) and its mutant derivative Origami B(DE3) as the host. Bioactive PalB was obtained in the reduced cytoplasm of BL21(DE3), implying that the formation of disulfide bond was not strictly required for functional expression. However, the expression was ineffective and was primarily limited by formation of PalB inclusion bodies and growth arrest, both of which were associated with PalB misfolding and deteriorated physiology. The culture performance in terms of cell growth and PalB expression level could be significantly improved by simultaneous coexpression of multiple chaperones of trigger factor and GroEL/ES, but not individual coexpression of either one of them. It was proposed that the two chaperones mediate the early stage and late stage of cytoplasmic PalB folding and would be required simultaneously for boosting both the overall PalB synthesis rate and the cytoplasmic folding efficiency. On the other hand, a much higher bioactive PalB was produced in Origami B(DE3) harboring the same PalB expression vector. Furthermore, the significant high bioactive PalB was produced by coexpression of periplasmic folding factor without a signal peptide (e.g., coexpression of DsbA, and DsbC). Coexpression of DsbA was found to be effective in enhancing PalB expression and such an improvement was more pronounced in Origami B(DE3), suggesting that both folding and disulfide bond formation could be the major factors limiting PalB expression. The fusion tag technique was also explored by constructing several PalB fusions for the evaluation of their expression performance. While the solubility was enhanced for most PalB fusions, only the DsbA tag was effective in boosting PalB activity possibly via both enhanced solubility and correct disulfide bond formation. Our results suggest that solubilization of PalB fusions did not necessarily result in the development of PalB activity which could be closely associated with correct disulfide bond formation. While PalB was stably expressed in the cytoplasm, most of the expressed gene product aggregated in cells as inactive inclusion bodies. In contrast, PalB was extremely unstable when expressed in the periplasm, also leading to poor expression performance. Such unstable PalB can be rescued by coexpression of several periplasmic folding factors, such as DegP, FkpA, DsbA, and DsbC, but not cytoplasmic chaperones. As a result, the performance for functional PalB expression in the periplasm was significantly improved. This is the first report demonstrating the use of folding factors to rescue the extremely unstable gene product that is otherwise completely degradable. On the other hand, functional expression of PalB in the periplasm was explored using four fusion tags, e.g., DsbC, DsbA, maltose binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags for functional expression of PalB, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of gene products, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegPS210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance for functional expression of PalB was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/liter. Without extracting protein from cells the whole cell can be directly used as a platform for the immobilized enzyme. Proof-of-concept experimentation was conducted by PalB display on the E. coli cell surface. By fusing the palB gene in between the signal peptide phoA and an autotransporter Protein EstA’s gene under the lac promoter, PalB was successfully displayed on the E. coli cell surface. However, cells encountered a severe physiological stress. Coexpression of various periplasmic folding factors, e.g., DegP, SurA, DsbA and DsbC could erease the physiological stress, but only DsbA was demonstrated to be effective to restore cell physiology and increase PalB expression level. Key words: enzyme, Escherichia coli, chaperone, folding factor, fusion tag, gene expression, Pseudozyma antarctica, lipase B, recombinant protein production

The L1 region of the porcine adenovirus (PAdV)-3 genome encodes a protein of 622 amino acids named IIIa. Although it binds a neighboring group of nine (GON) hexons at the capsid level and cement the icosahedral shell that contains the viral DNA, little is known regarding its function with respect to viral life cycle. Moreover, the known location of IIIa protein in the capsid may help to express targeting ligands for altering the tropism of PAdV-3. The objective of this study was to characterize the IIIa protein of porcine adenovirus Type 3 (PAdV-3). <p> In order to characterize the IIIa protein, polyclonal antisera were raised in rabbits against different regions of IIIa. Anti-IIIa sera detected a specific protein of 70 kDa in PAdV-3 infected cells using Western blot assay. Immunofluorescence studies indicated that IIIa is predominantly localized in the nucleus of PAdV-3 infected cells. Analysis of PAdV-3 IIIa using antibodies specific for N- and C- terminal domains of the protein suggested that although the N-terminus and C-terminal domains of IIIa are immunogenic, they are not exposed on the surface of PAdV-3 virions. These results were further confirmed by our inability to isolate a chimeric PAdV-3 virion containing a heterologous protein fused to the N-terminus or C-terminus of IIIa. <p>Functional analysis suggested that IIIa may transactivate the major late promoter and down regulate the early region (E) 1A promoter. In order to locate the domains of IIIa responsible for different functions, in-frame deleted/truncated forms of IIIa were constructed. Analysis of the deleted/truncated forms of IIIa suggested that a) the sequences located between amino acids 273-410 and between amino acids 410-622b) affect the nuclear localization and transactivation function respectively.<p>Since protein- protein interactions are important for the biological functions of the protein, we determined the interaction of PAdV-3 IIIa with other viral proteins. IIIa was found to interact with DNA binding protein (DBP), E3 13.7 kDa protein, hexon, fiber, and pIX. These results suggest that PAdV3 IIIa may do more in the viral life cycle than merely act as cement between the hexons to maintain capsid stability and may actually be involved in regulating early to late gene transcription at appropriate stages during viral infection.

Pasteurella multocida serotype A:3 is a gram-negative bacterial pathogen which is one of the causative agents of shipping fever in cattle. In this study, ompH and two fragments of plpE gene (plpEN and plpEC) were cloned from the genomic DNA of P. multocida P-1062 (ATCC 15743, serotype A:3) and plpEN-ompH and plpEC-ompH fusions were constructed. In vitro expression of the genes was shown in HEK-293 cells. Later, full-length plpE gene was cloned and the recombinant proteins were expressed in E. coli and purified. Three DNA vaccine formulations, namely pCMV-ompH, pCMV-plpEN-ompH and pCMV-plpEC-ompH and five recombinant protein based vaccines, PlpEN-OmpH, PlpEC-OmpH, OmpH, PlpEC and PlpE were generated. Recombinant proteins were formulated with at least one of the adjuvants: alum, CpG, alum-CpG, oil based and oil based-CpG. BALB/c mice were immunized with these vaccine formulations and their sera were used for the evaluation of antibody and serum IFN-&gamma / titers. Protective capacities of the vaccines were also evaluated via challenge of mice with 10 LD50 of P. multocida A:3. DNA vaccines induced immune responses, but did not provide protection. All protein vaccine formulations increased antibody levels and CpG containing formulations enhanced serum IFN-&gamma / titers. 100 &micro / g of PlpEC-OmpH protein adsorbed on alum adjuvant conferred 40% protection while no protection was obtained with PlpEN-OmpH. Next, the effects of CpG, or its alum and oil based combinations as adjuvants were investigated on PlpEC-OmpH mediated protection. The vaccine formulation composed of PlpEC-OmpH and oil based-CpG adjuvant conferred 100% protection. Finally, the mice were vaccinated with recombinant OmpH, PlpEC and PlpE formulated with oil based-CpG adjuvant. OmpH, PlpEC and PlpE formulations provided 50%, 60% and 100% protection, respectively. These findings implicated that recombinant PlpE and PlpEC-OmpH fusion proteins when formulated with oil based-CpG adjuvant are potent acellular vaccine formulation candidates against shipping fever.

The present study used Escherichia coli¡]BL21¡^that contained pET-23a-bpGH plasmids, to express black porgy growth hormone ¡]bpGH¡^. The bpGH was refolded at pH 11.3 in the presence of catalytic amounts of cysteine and purified by ion exchanger chromatography and gel filtration chromatography. The purified bpGH is a monomer and has a molecular weight of 22 kDa. Using the bpGH, the effects of the growth hormone on growth, essential amino acid deposition and nonspecific immunity in black porgy were studied. The experiment was a 4*3 ¡]diet*GH¡^ factorial design. Four experimental diets were formulated in that fish meal was replaced by the mixture of soya protein and gelatin so that fish meal / soya mixture = 100 / 0, 75 / 25, 50 / 50 and 25 / 75, respectively. GH treatments included non-injection, once per 3 days and once per 12 days. GH was injected intramuscularly at a dosage of 0.05 &#x00B5;g / g wet body weight. The growth trial lasted for 72 days. The results showed that GH administration significantly enhanced weight gain, feed efficiency, protein efficiency ratio and muscle methionine concentration of the fish. GH injection improved the growth performance of the fish fed low protein quality diets to a level equals to the groups fed high protein quality diets. These results indicate that GH injections enhanced the perferential absorption and deposition of the first limiting amino acid methionine from the diets. In addition, GH administration enhanced alternative complement activity and increased serum lysozyme concentration, implicating the enhancement of the immunity.

Thesis (M.S.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 42-44). Also available on the World Wide Web.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Elliot Chaikof; Committee Member: Marc Levenston; Committee Member: Robert Nerem; Committee Member: Vincent Conticello; Committee Member: Yadong Wang. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Recombinant antibodies and antibody fragments have become powerful tools for therapy, in vivo and in vitro diagnostics, and laboratory research. However, the production of antibody fragments in high yield for preclinical and clinical trials can be a serious bottleneck in drug discovery. This dissertation describes the development of novel screening systems for isolating antibody fragments and alternatively, E. coli genes, that facilitate expression in E. coli. In the first part of this work, we have developed a screening system for isolating Fab mutants exhibiting 4~5 fold higher expression level at 37oC compared to the parental Fab, by utilizing the APEx 2-hybrid system and multi-color FACS as a screening tool. In the APEx 2-hybrid system, the bacterial periplasm constitutes the milieu for the association of membrane-anchored bait protein and solubly expressed, epitope-tagged prey protein. Upon disruption of the outer membrane, only prey proteins that bind to the bait remain cell-associated and are detected by flow cytometry using fluorescently labeled anti-epitope antibodies. In the second part of this work we developed a new strategy to engineer scFv that can be expressed in soluble and active form in the absence of disulfide bonds. This was achieved using a strain incapable of forming disulfide bonds in proteins expressed in its periplasm in combination with the APEx 2-hybrid system. The selected clones exhibited higher solubility, activity, and stability than that of the wild type scFv in the reducing condition of the cytoplasm. Finally, we sought to isolate E. coli gene fragments that can enhance IgG production in the periplasm of E. coli by a newly developed screening system based on soluble expression of IgG and E. coli genomic fragments. The isolated gene fragments, which are located between moeA and iaaA in the E. coli chromosome, improved the total expression of polypeptides of IgG and also assembly of IgG. / text

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy