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The optimisation of recombinant protein production by animal cell cultures is important for the economic feasibility of these processes. Simultaneously with product yield, product authenticity is a crucial aspect to consider as it may per se affect the therapeutic value of such proteins. More defined culture media are being developed, particularly to ensure batch product consistency. A Chinese Hamster Ovary cell line (CHO 320) producing human interferon-γ (IFN-γ), a glycosylated protein, was chosen to investigate the effects of the culture environment on (I) cell growth, (2) product yield and (3) product authenticity. A statistical approach was used to identify important culture components for cell growth and IFN-γ production. When the concentration of the resulting positive variables was initially increased in culture, improvements of approximately 40% in both of these parameters were achieved; the glycosylation of IFN-γ was not affected. The former analysis also indicated that different stimuli were required for growth and production. Fed-batch feeding of glucose and glutamine, components depleted early from culture, did not prolong cell growth or IFN-γ production but the initial glycosylation pattern of IFN-γ was a function of glutamine concentration. Bovine serum albumin (BSA) was shown to have important role(s) in culture and cell growth was not possible in its absence. Pluronic F68, alone or in combination with a lipid mixture or linoleic acid, was able to restore cell growth in low BSA (1 mg/ml) cultures. However, IFN-γ production was significantly reduced and the extent of IFN-γ glycosylation also changed. These effects were related to: (1) BSA concentration, (2) BSA type, and ultimately, (3) lipid composition of the culture. The results reported in this thesis exhibit the necessity to consider the effects of the culture environment not only on cell growth and product yield but also on product authenticity throughout any optimisation process.

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This study of the relationship between heterologous protein production and bacterial growth has shown that foreign protein can account for as much as 30% of the total cell protein without the specific growth rate being affected. Higher levels of foreign protein production are achieved at lower growth rates. Foreign protein is synthesised without any increase in ribosome number or ribosome activity occurring in the recombinant cells relative to their parent strain, i.e. the capacity of parental and recombinant cells to synthesise protein is the same. These observations are explained in terms of a model in which Escherichia coli proteins are be divided into two types. Type I proteins are involved in the processes of transcription and translation. Type 2 proteins are those which serve other purposes. The model suggests that the synthesis of type 2 proteins will be decreased in order to accommodate foreign protein production. This implies that some Escherichia coli proteins are overproduced relative to the amount of that protein that is essential to the cell to maintain growth. Two dimensional electrophoresis showed that there was a decrease in some host cell protein levels between parental and recombinant cells. An increase in the levels of molecular chaperone proteins in recombinant cells grown under some conditions was observed. These data are explained in terms of our current understanding of the regulation of molecular chaperone production. The data obtained are discussed in relation to our current understanding of the growth of bacteria and the reported effects of high levels of foreign protein production on bacterial physiology.

In order to determine and study a protein structure, large amounts of it is needed. The easiest way to obtain a protein is to recombinantly overexpress it in the well-studied bacterium Escherichia coli. However, this expression host has one major disadvantage, overexpressed proteins might not be folded or be insoluble. Within the field of structural genomics, protein production has become one of the most challenging problems and the recombinant overexpression of viral proteins has in particular proven to be difficult. The first part of the thesis concerns the recombinant overexpression of troublesome proteins in E. coli. A method has been developed to screen for soluble overexpression in E. coli at the colony level, making it suitable for screening large gene collections. This method was used to successfully screen deletion libraries of difficult mammalian proteins as well as ORFeomes from five herpesviruses. As a result soluble expression of previously insoluble mammalian proteins was obtained as well as crystals of three proteins from two oncogenic human herpesviruses, all linked to DNA synthesis of the viral genome. The second part of the work presented concerns the structural studies of three herpesviral proteins. SOX from Kaposi’s sarcoma associated herpesvirus is involved in processing and maturation of the viral genome. Recently SOX has also been implicated in host shutoff at the mRNA level. With this structure, we propose a substrate binding site and a likely exonucleolytic mechanism. The holoenzyme ribonucleotide reductase is solely responsible for the production of deoxyribonucleotides and regulates the nucleotide pool of the cell. The small subunit, R2, has been solved from both Epstein Barr virus and KSHV. Both structures show disordered secondary structure elements in their apo-and mono metal forms, located close to the iron binding sites in similarity to the p53 induced R2 indicating that these two R2 proteins might play a similar and important role.

NK (natural killer) cells, with their ability to identify antigens and extraneous substances, available in the organism through various moleculary receptors, are an important component of the immune system. The NKR-P1A and NKR-P1B proteins belong to the lectin receptors of natural killer cells. Primary ligands of lectin receptors comprise terminal oligosaccharides of glycoproteins on the surface of target (e.g. tumor) cells. The interaction between carbohydrate structures on the surface of antigens and their binding partners on NK receptors is followed by triggering the effector function of NK cells against the targets. The NK cells and NK receptors findings and their interactions with ligands are greatly utilized in the treatment of cancer, viral and autoimmune diseases. Heterologous protein production in the eukaryotic organism brings a lot of advantages. Unlike the prokaryotic organism, the methylotrophic yeast Pichia pastoris has the capability of performing many posttranslational modifications resulting in production of biological active protein molecule. Usually, the P. pastoris expression system disposes of high level protein expression and is also generally regarded as being faster, easier, and less expensive to use than expression systems derived from other eukaryotes. In this thesis, I...

<p>Kinases and phosphatases are key regulatory proteins in the cell. The disruption of their activities leads ultimately to the abolishment of the homeostasis of the cell, and is frequently correlated with cancer. EphB1 is a member of the largest family of receptor tyrosine kinases. It is associated with neurogenesis, angiogenesis, and cancer. The cytosolic part of the human EphB1 receptor is composed of two domains. Successful generation of soluble constructs, using a novel random construct screening approach, led to the structure determination of the kinase domain of this receptor. The native structure and the complex structure with an ATP analogue revealed novel features in the regulation of the Eph family of kinases.</p><p>The structure of PTPA, an activator of protein phosphatase 2 A, a tumor suppressor and a key phosphatase in the cell was solved. The structure revealed a novel fold containing a conserved cleft predicted to be involved in interaction with PP2A.</p><p>Finally, the structure of YegS, an <i>Escherichia coli</i> protein annotated as a putative diacylglycerol kinase, has been determined. Beside the elucidation of its atomic structure, a phosphatidylglycerol (PG) kinase activity, never seen before, has been assigned to YegS based on biochemical studies. The YegS structure shows resemblance to the fold previously seen in NAD kinases. The structure also revealed the existence of a novel metal site that could potentially play a regulatory role. The YegS structure has important implications for understanding related proteins in pathogenic organisms and is the first homologue of a human lipid kinase for which the structure has been elucidated.</p>

The production of recombinant glycoproteins from mammalian cell cultures requires robust processes that can achieve high protein yield while ensuring the efficacy of these proteins as human therapeutics. We describe two approaches currently being developed in our group to genetically engineer cell lines with desirable characteristics for recombinant protein production. To enhance the degree of sialylation in the glycoprotein product, we propose to increase intracellular sialic acid availability by overexpressing the CMP-sialic acid transporters. We are also interested in engineering mammalian cells that can proliferate at reduced cultivation temperatures. Low temperature cultivation of mammalian cells has been shown to enhance glycoprotein production but reduces cell growth. It is hypothesized that a mutant cell line that can proliferate at low temperatures may be coupled with low temperature cultivation to improve recombinant protein production. / Singapore-MIT Alliance (SMA)