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Minimising the time from 'scientific breakthrough'to clinical trial of a 'drug candidate' protein is a critical component leading to a successful product release. Crystallographic characterisation has become a standard requirement prior to clinical trial requiring milligram quantities of protein. The optimisation of protein expression systems is therefore of great commercial and social importance and represents a significant technical challenge. Without it, making enough protein for crystallography can quite literally take years. Baculoviral expression of recombinant protein by infection of an insect cell host is a well established technique in modern biotechnology. Although a limit to recombinant protein production in batch culture exists the mechanism has not been demonstrated. In particular, there has been no discussion of how biomass accumulation kinetics relate to the system limits in terms of final recombinant protein yield. The central aim of this thesis was therefore to quantitatively account for the dynamic behaviour in macromolecular compartments after baculovirus infection of insect cells, the rationale being that a rudimentary level of mechanistic structure can greatly enhance our ability to capture transient behaviour. The catalytic mass dictates the rate of total biomass accumulation in the baculovirus expression vector system (BEVS) and is directly proportional to the total RNA content of both baculovirus infected and uninfected Sf9 cells. During infection the total RNA concentration reaches a catalytic limit causing a switch from exponential to zero order mass accumulation kinetics. Importantly, this extends to individual cells as confirmed using a population balance model for the cell volume distribution after the switch to linear growth. By flow cytometry, a positive correlation between RNA content and cell size post infection validated this modelling assumption. The rate of mass accumulation slows down during the first 12 hours post infection (hpi). This is consistent with the decrease in both specific consumption rates of glucose and oxygen when using cell mass rather than cell number as a basis. A decrease in the geometric standard deviation (óg) of the cell volume distribution during the first 12 hpi indicates that cells enter the lower growth rate at times inversely proportional to their volume. Using several approaches no obvious biological mechanism to account for the empirical model was identified. The use of óg kinetics provides a novel tool for characterising the relative behaviour of infected cells in the BEVS. The effect of multiplicity of infection (MOI) on virus timing events between cultures was also tested. Little or no effect of MOI was observed on the timing of virus induced events during synchronous infections. The óg kinetics did indicate virus events occur 5 hours earlier at a MOI of 100 compared to a MOI of 20 plaque forming units per cell. There was however, no significant evidence of earlier death kinetics when measured using Trypan blue dye exclusion to measure cell membrane integrity. Virtually no effect of MOI on virus timing was observed using â-galactosidase production profiles. The viral DNA mass (vDNA) was measured using real time quantitative PCR (RTQ PCR) and has a doubling time of 2 hours. A vDNA template limited replication model fit the data well. Viral replication proceeds from 6 until 24 hpi with the average infected cell accumulating between 12 000 and 84 000 vDNA copies when replication stops. In theory, a dynamic equilibrium could have been present after the commencement of virus budding but this was not the case. At least 62% of the total DNA increase post infection is viral. No more than 16% of the total vDNA produced actually bud from the infected cell. This overproduction of vDNA is probably due to the wild type history of the virus, which normally occludes virions in a crystalline polyhedrin matrix within the cell nucleus as part of its life cycle. The approach taken here provides a framework for characterisation of both viral and total mass accumulation with the use of a few simple intracellular macromolecular pools. This thesis demonstrates that the BEVS limit in batch culture is not simply a result of the exhaustion of an amino acid using a case study of amino acid consumption by uninfected Sf9 cells for a 300 hour culture period. Future attempts to identify the system limits and will require the linkage of a mechanistic model with a more extensive and accurate analysis of important metabolites and specific intracellular species.

Relaxin is a small peptide hormone that has demonstrated potential therapeutic actions for cardiovascular disease and fibrosis. Additionally, relaxin has demonstrated the ability to protect the heart from injuries caused by ischemia and reperfusion, promote the healing of ischemic ulcers, and counteract allergic responses. The objective of this research was to express fully processed porcine relaxin in transgenic tobacco plants, as an alternative to current methods of producing relaxin. Two recombinant relaxin genes were constructed that contained the patatin signal peptide cDNA fused in frame to prorelaxin cDNA, which was codon-optimized for expression in Nicotiana tabacum, under the control of either the "super" promoter or the dual enhanced cauliflower mosaic virus 35S promoter. Eighteen transgenic tobacco plants were generated that were transformed with the above recombinant genes. Preprorelaxin, mRNA was detected in 12 of the transgenic plants. Fully processed relaxin protein was not found in any tobacco plants that had demonstrated gene expression by northern blot analysis. Preprorelaxin was only identified in extracts from transgenic plants that contained the insoluble protein fraction, as determined by western blot analysis. Additionally, an increased yield of preprorelaxin was identified after incubation of tobacco leaves in an ubiquitin inhibitor. / Master of Science

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No description available.

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.

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.

Hepatocyte growth factor (HGF) is a multifunctional protein, which secrets via Golgi complex after synthesized, and is hydrolyzed into an active heterodimer containing an £\ and a £] chain by extracellular protease. It is known that HGF functions through surface domain of Met, and thus induces mitosis and metastasis. The interaction domain of HGF is believed to be located in the £\-chain. In order to study these findings structurally and functionally, we designed and constructed four different recombinant coding regions of the gene (NK1, NK2, NK3, and NK4) which was then successfully expressed in E. coli. Purification of these four different recombinant proteins with glutathione-agarose column showed that all of the four constructs had been successfully expressed with some degradations. Cell proliferation assay showed that the recombinant proteins inhibited the growth of breast cancer cells to some extent. The assay also showed that GST-NK1 and GST-NK2 were better inhibitors than GST-NK3 and GST-NK4 to the cancer cells. It is concluded that E. coli expression is an appropriate system for achieving functional HGF.

Neutrophils ordinarily infiltrate the female reproductive tract subsequent to mating or artificial insemination, resulting in reduced fertility. Recently, it was demonstrated that equine neutrophil extracellular traps (NETs) entangled sperm in these DNA-rich structures, interfering with their normal transport through the female reproductive tract. Seminal plasma (SP) or proteinaceous extracts from SP inhibited sperm-neutrophil binding and specifically degraded sperm-activated NETs, without suppressing bactericidal activity of neutrophils. Fertility-associated antigen (FAA), a 31 kDa naturally occurring heparin-binding protein (HBP) produced by the accessory sex glands, has been shown to bind to sperm and potentiate heparin-induced capacitation. FAA shares 87% identity with DNase I-like family members, and contains two internal DNase-I-like peptide motifs. The purpose of this study was to determine if a recombinant form of FAA displayed capacitating effects associated with the native protein and to determine whether rFAA displayed DNase activity similar to SP or SP protein extracts to inhibit sperm-neutrophil binding.