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There are an estimated 350 million people chronically infected with Hepatitis B Virus (HBV), of which approximately 600 000 die each year from HBV complications including cirrhosis and liver cancer. The X protein from HBV (HBx) has been implicated in the progression of chronic HBV to liver cancer and has been reported to manipulate several critical cellular pathways. These include the cell cycle, the tumour suppressor protein p53, protein degradation and signal transduction pathways. The role of these interactions in HBV replication and the viral lifecycle is currently unknown. The lack of animal models and infectable cell lines together with solubility and stability issues related to the HBx protein have made progress difficult. The reliance on approximate cellular and animal models has yielded many discordant studies that have confounded our interpretations of the role of HBx. There have been no novel approaches attempting to express HBx at a quantity and quality sufficient for high resolution X-ray and nuclear magnetic resonance structural determination. Additionally no bioinformatic analyses have been applied to HBx, and thus distinctive features of HBx that may be responsible for these challenges have not been reported. This thesis describes the detailed experimentation to express and purify HBx in a functional, soluble and stable form. The study focussed on Saccharomyces cerevisiae and Semliki Forest Virus (SFV) expression systems, together with the use of a solubility-enhancing Maltose Binding Protein protein tag (MBP). The S. cerevisiae-based pYES2 and YEp and mammalian expression vectors showed production of HBx protein. However HBx that had been expressed using S. cerevisiae and human cells could not be reliably detected in Western blots using antibodies raised against E. coliexpressed HBx. This result was despite the positive visualisation of HBx using the same antibodies and immunofluorescence microscopy. This validated previous reports describing the variable antigenicity of HBx. Furthermore these findings supported the decision to develop eukaryotic-based HBx expression vectors as results suggested structural differences between eukaryote and prokaryote expressed protein. HBx was subsequently detected and purified in a soluble and active form using an MBP tag as well as a SFV expression vector. All of these options provide an excellent point from which further work at optimising HBx expression and structural elucidation can occur. Bioinformatic analysis of HBx suggested the presence of protein disorder and protease sensitive sites within the negative regulatory domain of HBx. Literature descriptions of the molecular promiscuity that protein disorder allows, offers an explanation for the presence of the discordant findings on HBx interactions and functions. It is generally accepted that proteins containing disorder are tightly regulated and thus experimental systems employing overexpression methodologies may encourage cellular toxicity and non-specific interactions through the use of short linear motifs. Evolutionary analysis of HBx sequences revealed that the eight HBV genotypes (A-H) showed concordance regarding synonymous and non-synonymous substitutions at the overlapping and non-overlapping domains of hbx. Substitutions in hbx were most common at positions where a synonymous substitution occurred in the overlapping partner gene. The presence of sites under positive, neutral and negative selection were identified across the length of HBx. The different genotypes showed positive selection indicating selective pressures unique to each, thus offering a contributing explanation for the variable disease severity observed between the subtypes. Overall, this thesis has provided novel methods to express and purify HBx in S. cerevisiae and mammalian cells. These methods, together with an increased understanding of the nature of HBx sequences through bioinformatic analysis, pave the way to conduct both structural studies and biological assays to elucidate the genuine roles of HBx in the HBV lifecycle and its contribution to the progression to liver cancer.

Functional biomaterials are being developed as scaffolds to support endogenous cells and to promote the regeneration of ischemic tissue. The aim for this study was to develop a new translational platform for injectable hydrogels using recombinant human collagen (rHC) of two types: type I (TI) and type III (TIII). The collagen solutions were characterized to ensure batch-to-batch consistency and protein integrity. The hydrogel preparation protocol was extensively monitored to ensure ease of use and high-quality production. Post-gelation, rHC TIII have a higher viscosity compared to rHC TI, yet water content was high for both hydrogels. The cross-linking degree is similar for both rHC hydrogels, which are stable well above physiological temperatures, but rHC TI is more susceptible to enzymatic degradation than rHC TIII. Furthermore, the micro-architecture differed with pore size dimensions of rHC TIII being significantly larger than that of rHC TI. Cardiac fibroblasts were cultured on the rHC hydrogels, and cells attached readily to the scaffold environment, which promoted proliferation. The rHC matrices mechanical and biological properties provide structural support, and demonstrate biodegradability and biocompatibility. The intrinsic physical differences between the rHC hydrogels will likely have implications in future studies. In conclusion, the rHC TI and TIII hydrogels are proven to be suitable matrices for continued investigation towards future translational applications.

Poon, Chi-to, Geoffrey. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 115-125). / Acknowledgements --- p.I / Abbreviations --- p.II / Abstract --- p.III / Table of content --- p.IV / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Importance of growth enhancement in aquaculture --- p.1 / Chapter 1.2 --- Physiological effect of growth hormone --- p.1 / Chapter 1.3 --- Progress in teleost growth hormone research --- p.3 / Chapter 1.4 --- Grass carp and its aquaculture --- p.5 / Chapter 1.5 --- Route of administration of growth hormone --- p.8 / Chapter 1.6 --- Nomenclature of baculovirus --- p.9 / Chapter 1.7 --- Biology of baculovirus --- p.10 / Chapter 1.8 --- Control of gene expression of virus-infected cells --- p.13 / Chapter 1.9 --- Theme of the thesis --- p.14 / Chapter Chapter 2 --- Materials and Methods --- p.18 / Chapter 2.1 --- Synthesis and purification of primers --- p.18 / Chapter 2.2 --- Modification of gcGH cDNA by polymerase chain reaction (PCR) --- p.20 / Chapter 2.3 --- TA cloning of PCR product --- p.20 / Chapter 2.4 --- Purification ofDNA fragment from agarose gel by GENECLEAN´ёØ --- p.21 / Chapter 2.5 --- Recovery of low molecular weight DNA fragment from agarose gel --- p.22 / Chapter 2.6 --- Small scale preparation of plasmid DNA --- p.23 / Chapter 2.7 --- Large scale plasmid preparation by QIAGEN´ёØ --- p.24 / Chapter 2.8 --- Preparation of competent Escherichia coli JM109 for transformation --- p.25 / Chapter 2.9 --- Transformation of plasmid into competent Escherichi coli JM109 --- p.26 / Chapter 2.10 --- Cell culture of BmN cell line --- p.26 / Chapter 2.10.1 --- Preparation of TC-100 insect medium --- p.27 / Chapter 2.10.2 --- Preparation of Grace's medium --- p.27 / Chapter 2.11 --- Extraction of wild-type Bombyx mori nuclear polyhedrosis virus DNA --- p.28 / Chapter 2.12 --- Transfection of BmN cells with Bombyx mori nuclear polyhedrosis virus DNA by DOTAP´ёØ --- p.28 / Chapter 2.13 --- Agarose plaque assay --- p.29 / Chapter 2.14 --- Lifting of vius plaque onto nitrocellulose filter paper --- p.30 / Chapter 2.15 --- Synthesis of radiolabelled DNA probe --- p.31 / Chapter 2.16 --- Pre-hybridization and hybridization of recombinant virus DNA on nitrocellulose paper --- p.31 / Chapter 2.17 --- Purification of recombinant virus by dot-blot manifold --- p.33 / Chapter 2.18 --- Preparation of cell lysate from virus-infected BmN cells --- p.33 / Chapter 2.19 --- Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.34 / Chapter 2.19.1 --- Staining of the gel by Coomassie blue method --- p.35 / Chapter 2.19.2 --- Staining of the gel by silver staining method --- p.35 / Chapter 2.20 --- Determination of protein concentration by Bradford's method --- p.36 / Chapter 2.21 --- Determination of total protein concentration by Folin-Lowry method --- p.36 / Chapter 2.22 --- Detection of grass carp growth hormone by Western blotting --- p.37 / Chapter 2.23 --- Preparation of native recombinant grass carp growth hormone for iodination --- p.38 / Chapter 2.24 --- Iodination of recombinat grass carp growth hormone by IODO-GEN´ёØ --- p.38 / Chapter 2.25 --- Purification of radiolabelled recombinant grass carp growth hormone --- p.39 / Chapter 2.26 --- Radioimmunoassay (RIA) for detection of recombinant grass carp growth hormone --- p.40 / Chapter 2.27 --- Ammonium sulphate precipitation --- p.41 / Chapter Chapter 3 --- Vector Construction --- p.42 / Chapter 3.1 --- Components of parent vector pBM030 --- p.42 / Chapter 3.2 --- Construction of pBM-EE --- p.44 / Chapter 3.3 --- Constrcution of pBM-EX --- p.47 / Chapter Chapter 4 --- Results --- p.51 / Chapter 4.1 --- Construction and purfication of recombinant baculovirus --- p.51 / Chapter 4.2 --- Expression of recombinant grass carp growth hormone in BmN cells --- p.55 / Chapter 4.3 --- Expression of recombinant grass carp growth hormone in Bombyx mori larva --- p.62 / Chapter 4.4 --- Putative physical characteristics of the recombinant grass carp growth hormone --- p.67 / Chapter 4.5 --- Purification of the grass carp growth hormone in Bombyx mori larva --- p.69 / Chapter 4.5.1 --- Ammonium sulphate precipitation --- p.69 / Chapter 4.5.2 --- Gel filtration --- p.72 / Chapter 4.5.3 --- Hydrophobic interaction chromatography --- p.75 / Chapter 4.5.4 --- Anion exchange chromatography --- p.78 / Chapter 4.5.5 --- Reverse phase chromatography --- p.90 / Chapter Chapter 5 --- Discussions --- p.99 / Chapter 5.1 --- Merits of baculovirus expression system against other expression systems --- p.99 / Chapter 5.2 --- Basic design of the recombinant baculovirus transfer vector --- p.100 / Chapter 5.3 --- Potential for Mutation of the Baculovirus during Homologous Recombination --- p.101 / Chapter 5.4 --- Cleavage of Signal Peptide from the Expressed Protein --- p.103 / Chapter 5.5 --- Difference in recombinant gcGH expression levelin EE4-7 and EX3-16 --- p.103 / Chapter 5.6 --- Purification of recombinant gcGH protein --- p.106 / Chapter 5.6.1 --- Chromatographic behaviour of recombinant gcGH in Q-Sepharose column --- p.106 / Chapter 5.6.2 --- Problem of aggregation of recombinant gcGH --- p.107 / Chapter 5.6.3 --- Solvent system used in recombinant gcGH purification --- p.108 / Chapter 5.6.4 --- Protein denaturating effect of the solvent system --- p.109 / Chapter 5.6.5 --- Protein yield --- p.110 / Chapter 5.7 --- Problems and accuracy of radioimmunoassay --- p.110 / Chapter Chapter 6 --- Further study --- p.113 / Chapter Chapter 7 --- References --- p.115 / Appendix I --- p.126 / Appendix II: Construction of the Supervector --- p.127

by Sharon, Chui-Wah Luk. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 128-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Transcriptional regulation is a determinant of stability of recombinant protein production in CHO cells. Fundamental studies of recombinant gene transcription in relation to chromatin environment and promoter regulation are important for CHO cell line development and selection. This study has developed a methodology based on a cell/vector system to study recombinant transcription and expression stability of different promoters and/or proteins in the similar genomic environment. The CHO-FRT mini-pools developed in this project were mini-pools of CHO-S cell lines containing Flp Recombination Target (FRT) sites with ß-galactosidase gene, under the influence of a SV40 promoter. Continuous culture of these mini-pools for 8 weeks using a robotic system demonstrated that 20% of the mini-pools studied revealed an unstable profile (with 30% loss of protein expression). Two of these mini-pools with different characteristics, CHO-FRT 1 (low producer/unstable) and CHO-FRT 108 (high producer/stable), were selected to be used on the study of influence of SV40 and CMV promoters in long-term recombinant expression. Genes encoding fluorescent proteins were integrated in a site-directed manner under the influence of SV40 or CMV promoters. A sub-clonal population of the top 10% yellow fluorescent protein (YFP) expressing cells of each mini-pool/promoter combination was selected by cell sorting and cultured for 4 weeks. During this period protein expression was monitored by flow cytometry and compared between both promoters. The results revealed that both SV40 and CMV promoters had an unstable expression with different degrees of instability and long-term expressing behaviours. For CMV, instability was considerably high displaying a long-term logarithmic loss of 50-80% of productivity while for SV40 the loss of productivity observed was only 40-45% with a linear behaviour during long-term culture. The vector system generated contained an MS2-RNA tag sequence cloned 3'- of the recombinant gene to track the recombinant mRNA by using the MS2/MCP-GFP system. This study showed the development of a protocol to measure the transcriptional output of recombinant promoters in CHO cells. The results showed background signal in CHO cells that requires further optimisation studies to allow the direct live cell image quantification of the transcriptional activity of recombinant promoters. Although not yet optimised, the successful combination of site-directed integration with recombinant mRNA tagging method has the potential to become a valuable tool to study the mechanisms of transcriptional activity and stability of transcription driven by different promoters in CHO cells.

Introduction: Mammalian cell recombinant bone morphogenetic protein (rBMP) synthesis is reported to be poor. The BMP pro-domain may be involved in folding, stability and secretion. Objectives: Investigate the effect of inhibition of pro-domain cleavage on rhBMP-2 production. Methods: CHO cells transfected with human BMP-2 (hBMP-2) were cultured in the presence of the proprotein convertase inhibitor 9DR in short (multi-well) and long-term (bioreactor) cultures. Mature and proBMP secretion was measured by ELISA and characterized by Western blot. BMP activity was determined by C2C12 bioassay. Results: 9DR significantly enhanced the yields of both pro- and mature hBMP-2 in short and long-term cultures, without any negative effects on cell growth or viability. The rhBMP-2 was biologically active. ProBMP-2 could be converted by exogenous furin treatment into mature BMP-2 as shown by Western blot. Conclusions: 9DR increases rhBMP-2 production and is a simple, but effective way to enhance the yield of active, mature BMP-2 in CHO cells.

Introduction: Mammalian cell recombinant bone morphogenetic protein (rBMP) synthesis is reported to be poor. The BMP pro-domain may be involved in folding, stability and secretion. Objectives: Investigate the effect of inhibition of pro-domain cleavage on rhBMP-2 production. Methods: CHO cells transfected with human BMP-2 (hBMP-2) were cultured in the presence of the proprotein convertase inhibitor 9DR in short (multi-well) and long-term (bioreactor) cultures. Mature and proBMP secretion was measured by ELISA and characterized by Western blot. BMP activity was determined by C2C12 bioassay. Results: 9DR significantly enhanced the yields of both pro- and mature hBMP-2 in short and long-term cultures, without any negative effects on cell growth or viability. The rhBMP-2 was biologically active. ProBMP-2 could be converted by exogenous furin treatment into mature BMP-2 as shown by Western blot. Conclusions: 9DR increases rhBMP-2 production and is a simple, but effective way to enhance the yield of active, mature BMP-2 in CHO cells.

Computational models of recombinant production of tissue-type Plasminogen Activator (tPA) were created, studied and compared for two hosts, Chinese Hamster Ovary (CHO)cells and Escherichia coli (E. coli), using SuperPro® Designer. In addition, several fermentations were run using enhanced Yellow Fluorescent Protein (eYFP) in E. coli to provide knowledge for the SuperPro model and to explore the effect of temperature when used to maintain dissolved oxygen in a high density fed-batch fermentation. The models show that production of tPA is feasible using either host, but under the current basecase CHO holds the economic advantage despite the initial higher capital costs. In order to become more competitive with CHO, production using E. coli must become higher on a cell specific level and the potential of refolding insoluble protein in inclusion bodies should be explored. Since E. coli’s growth rate allows for higher plant throughput in a given production year, if this was combined with strains which produce higher titers of protein than those available in literature, it would allow E. coli to become competitive with CHO for the production of recombinant tPA. Experiments demonstrate that temperature control can be used to slow the metabolic rate of E. coli, allowing aerobic conditions to be maintained in the high density fermentations. Although temperature reduction has also been used to increase the yield of soluble protein, it is likely this occurs with reduced protein production. Temperature control was initiated using five minute moving averages to monitor overall oxygen and stirrer speed trends. Temperature was dropped 5 °C when averaged oxygen content fell below 18% and averaged stirrer speeds were greater than 1000 rpm. Temperature controlled runs for E. coli BL21DE3 producing eYFP appeared to allow the cultures to maintain better aerobic conditions. It is known that eYFP was produced since homogenized cell paste fluoresced yellow under UV light. However, protein analysis was hampered due to low protein production even after induction. Purifications involving large amounts of cell paste (50 g or more) were difficult to perform and all purificitiatons resulted in contamination by other proteins. Several recommendations can be made. The modeling would be greatly facilitated by additional information such as equipment specifications at large-scale production. The work with eYFP containing E. coli would be greatly enhanced by better strain selection. Choosing strains which over-express the protein of interest on the small scale would lead to better results in the fermentor. A densiometric analysis of the SDS PAGE gels run would allow a better understanding of general proteomic response to temperature control. When combined with mass spectrometry this may lead to different approaches in reducing temperature. Temperature control is often thought to increase soluble protein. From the densiometric SDS PAGE analysis of both the supernatant and pellet after homogenization it would be interesting to examine the partioning of recombinant protein into soluble and insoluble forms in future experiments.

Computational models of recombinant production of tissue-type Plasminogen Activator (tPA) were created, studied and compared for two hosts, Chinese Hamster Ovary (CHO)cells and Escherichia coli (E. coli), using SuperPro® Designer. In addition, several fermentations were run using enhanced Yellow Fluorescent Protein (eYFP) in E. coli to provide knowledge for the SuperPro model and to explore the effect of temperature when used to maintain dissolved oxygen in a high density fed-batch fermentation. The models show that production of tPA is feasible using either host, but under the current basecase CHO holds the economic advantage despite the initial higher capital costs. In order to become more competitive with CHO, production using E. coli must become higher on a cell specific level and the potential of refolding insoluble protein in inclusion bodies should be explored. Since E. coli’s growth rate allows for higher plant throughput in a given production year, if this was combined with strains which produce higher titers of protein than those available in literature, it would allow E. coli to become competitive with CHO for the production of recombinant tPA. Experiments demonstrate that temperature control can be used to slow the metabolic rate of E. coli, allowing aerobic conditions to be maintained in the high density fermentations. Although temperature reduction has also been used to increase the yield of soluble protein, it is likely this occurs with reduced protein production. Temperature control was initiated using five minute moving averages to monitor overall oxygen and stirrer speed trends. Temperature was dropped 5 °C when averaged oxygen content fell below 18% and averaged stirrer speeds were greater than 1000 rpm. Temperature controlled runs for E. coli BL21DE3 producing eYFP appeared to allow the cultures to maintain better aerobic conditions. It is known that eYFP was produced since homogenized cell paste fluoresced yellow under UV light. However, protein analysis was hampered due to low protein production even after induction. Purifications involving large amounts of cell paste (50 g or more) were difficult to perform and all purificitiatons resulted in contamination by other proteins. Several recommendations can be made. The modeling would be greatly facilitated by additional information such as equipment specifications at large-scale production. The work with eYFP containing E. coli would be greatly enhanced by better strain selection. Choosing strains which over-express the protein of interest on the small scale would lead to better results in the fermentor. A densiometric analysis of the SDS PAGE gels run would allow a better understanding of general proteomic response to temperature control. When combined with mass spectrometry this may lead to different approaches in reducing temperature. Temperature control is often thought to increase soluble protein. From the densiometric SDS PAGE analysis of both the supernatant and pellet after homogenization it would be interesting to examine the partioning of recombinant protein into soluble and insoluble forms in future experiments.