The purity of heterologous recombinant proteins is of utmost importance to the pharmaceutical sector since most are consumed as therapeutic agents by humans. Variability caused by co- and posttranslational modifications is a major concern in pharmaceutical production. In order to develop strategies which guarantee a homogeneous product in a robust production process, it is important to better understand the metabolic basis of the synthesis of related non-canonical amino acids. So far, studies have identified high glucose fluxes in connection to oxygen limitation and overexpression of leucine-rich proteins as possible reasons for the production of non-canonical amino acids and their incorporation into heterologous proteins expressed in Escherichia coli. The results presented in this work provide evidence that oscillations in the concentrations of glucose and oxygen as they occur in inhomogeneous industrial scale bioreactors potentiate the synthesis and incorporation of norvaline into the leucine-rich protein IL-2, heterologously expressed in E. coli W3110M, as observed in well-mixed homogenous cultures and perturbed shake flask cultivations. In order to represent the heterogeneities existing in large-scale bioreactors, two experimental setups were applied, using a simple shake flask scale-down model developed to monitor dissolved oxygen and pH online during a batch and fed-batch cultivation phases. Results here show that by applying repeated glucose pulses to the glucose limited culture, which consequently induce oscillations in dissolved oxygen, norvaline is accumulated. Analysis of inclusion bodies that resulted from the expressed IL-2 revealed the presence of norvaline in the protein. A higher concentration of norvaline was observed in the oscillating scale-down model compared to the non-perturbed culture, which suggests that the conditions as they typically occur in large scale bioreactors may be critical for product quality. The results and tools, developed in this work are a solid basis for future cell engineering approaches to overcome the challenges in view of product quality.

Cotransin is a cyclodepsipeptide inhibitor of protein translocation and has been demonstrated to inhibit cotranslational translocation of a variety of proteins by targeting the mammalian ER translocation channel Sec61 (Besemer, Harant et al. 2005, Garrison, Kunkel et al. 2005). Genetic screens in cancer cells found out the mutations near the luminal plug domain of Sec61 confer resistance to cotransin inhibition and thus outline the proposed binding site for cotransin (MacKinnon, Paavilainen et al. 2014). However molecular details of cotransin interactions remain unknown. Purpose of my first project was to express the human Sec61 translocation channel in correct stoichiometric ratios. To our knowledge, heterotrimeric expression of Sec61 has not been achieved previously. Baculovirus system was chosen express the Sec61 heterotrimeric complex. To vary expression levels of Sec61α and Sec61γ relative to Sec61β, separate baculovirus constructs were prepared. Proper co-transfection ratios between these viruses to express Sec61subunits in correct stoichiometric ratios were calculated during my pro gradu and insect cell expression was then scaled up using the determined virus ratios. Results from Sec61 expression have returned sufficient quantities of the translocation channel for biochemical analyses. Final expression seems to contain high lipid/protein ratio, which may have been caused due to insect cells not-fully adapted to the media. The expression should be repeated in well-adapted healthy insect cells and then accessed for protein quality. For its isolation for crystallization studies, co-immunoprecipitation may be a preferred way to pull down the Sec61 complex. Detergent based solubilization may be alternatively used to isolate Sec61. The second part of my pro gradu work included expression of SecYEβ translocation channel from Pyrococcus furiosus, along with its various humanized mutants, whose DNA constructs had been provided by Dr. Ville Paavilainen. A major goal of this work was to express the mutants which bind to cotransin. Via photo-crosslinking and click chemistry analyses, a mutant binding to cotransin was identified and scaled-up. Our photo-crosslinking studies were able to demonstrate that the wild-type SecYEβ does not bind to cotransin in vitro. Results from photo-crosslinking assays during this project also demonstrated that other known translocation inhibitors Mycolactone and Apratoxin A can bind to mammalian Sec61 channel. These results are consistent with unpublished work from Paavilainen lab (Paatero et al).

Bacterial proteins DsbB, VKOR, and the mammalian protein VKORC1 share similar functions involving electron transfer processes. While DsbB is not homologous to the bacterial and mammalian VKOR proteins, the three proteins share overall structural features. Based on the similarities between the three proteins and the finding that mtbVKOR can replace DsbB in E. coli, we considered the possibility of rat VKORC1 displaying similar functionality as mtbVKOR. Genetic selection and screening was done on an EMS mutagenized ΔdsbB strain expressing rat VKORC1wt from a plasmid for isolation of E.coli mutants that would facilitate complementation of the lack of DsbB by VKORC1wt. The principle for the genetic selection and screening is the restoration of disulfide bond forming pathway by replacement of DsbB. This phenotype of complementation can be assayed by restoration of motility, resistance to TCEP, and β-galactosidase inactivation on ∆dsbB strain. Results of the selection and screening process revealed mutations in the VKORC1 gene instead of the E.coli chromosome. On the other hand, we used a rational approach other than genetic screening. This approach involved targeting YidC hydrophilic groove, previously identified upon selection of mutants that facilitated functional expression of VKORC1∆AAR; a deletion of amino acids 31–33 (AAR), where other mutations inactivating protease HslV were also identified. For this approach, chromosomal mutations were introduced on selected residues in the YidC hydrophilic groove then functional expression of VKORC1wt or enhanced expression of VKORC1∆AAR in the new strains was assayed. We identified novel YidC mutants enhancing the expression of VKORC1∆AAR. From the analysis of these mutations and the VKORC1 mutations obtained from the screens, we concluded that the charge imbalance by VKORC1wt violates the positive-inside rule impeding its ability to substitute DsbB. The correct assembly of VKORC1∆AAR provided insight on the involvement of E.coli YidC in correct folding and insertion of foreign membrane proteins, with the hydrophilic groove being core for its membrane insertase functions. The improved functional expression of VKORC1∆AAR upon HslV inactivation provided insight on the mis-assembly of foreign membrane proteins as a quality control system. These findings suggested that the native VKORC1wt may not assemble properly in the E.coli inner membrane, and is degraded by proteases as a quality control mechanism.

Enzymic hydrolysis of cod fish waste was investigated using two enzymes (trypsin and bromelain). A fish protein hydrolysate (FPH) powder and frozen flake hydrolysate were produced using a spray drier and an ice flake machine. Functional properties of the FPH were assessed with respect to the molecular weight (MW) spectrum. The characteristics of solubility and emulsification for the hydrolysate showed it to be suitable for use as a binder compared with egg albumin (EA) and soy bean isolate (ISB) for fish products. Fishburgers with improved texture, succulence and reasonable cooking losses were made successfully from cod fish mince incorporating a vegetable oil/water emulsion stabilised by FPH. Taste panels were carried out and overall acceptability of the fishburgers made from the FPH emulsion was better than fishburgers containing EA and ISB emulsions. Economic evaluation and specification of a pilot plant were done for both FPH powder and frozen flake hydrolysate production. This work strongly emphasises that FPH should only be used in fish products which need a good binder and where the flavour/taint problems of use in other products, e.g. beverages, pasta, will not arise.

A powdered product that was light in colour and had no off flavour or odour was made by the separation, ultrafiltration and spray drying of bovine blood. To establish the variability of the production methods samples were taken at stages during processing. A one year stability trial on one batch of powder was also done. Samples were analysed to indicate their physical characteristics, approximate composition and microbiological content. Model systems were established and used to assess the functional properties of the powder. Ten bovine plasma powders were produced with little indication of variation between batches. The powder was stable.

abstract: Protein crystallization has become an extremely important tool in biochemistry since the first structure of the protein Myoglobin was solved in 1958. Survival of motor neuron protein has proved to be an elusive target in regards to producing crystals of sufficient quality for X-ray diffraction. One form of Survival of motor neuron protein has been found to be a cause of the disease Spinal Muscular Atrophy that currently affects 1 in 6000 live births. The production, purification and crystallization of Survival of motor neuron protein are detailed. The Fenna-Matthews-Olson (FMO) protein from Pelodictyon phaeum is responsible for the transfer of energy from the chlorosome complex to the reaction center of the bacteria. The three-dimensional structure of the protein has been solved to a resolution of 2.0Å with the Rwork and Rfree values being 16.6% and 19.9% respectively. This new structure is compared to the FMO protein structures of Prosthecocholoris aestuarii 2K and Chlorobium tepidum. The early structures of FMO contained seven bacteriochlorophyll-a (BChl) molecules but the recent discovery that there is an eighth BChl molecule in Ptc. aestuarii 2K and Cbl. tepidum and now in Pld. phaeum requires that the energy transfer mechanism be reexamined. Simulated spectra are fitted to the experimental optical spectra to determine how the BChl molecules transfer energy through the protein. The inclusion of the eighth BChl molecule within these simulations may have an impact on how energy transfer through FMO can be described. In conclusion, a reliable method of purifying and crystallizing the SMNWT protein is detailed, the placement of the 8th BChl-a within the electron density and the implications on energy transfer within the FMO protein when the 8th BChl-a is included from the green sulfur bacteria Pld. phaeum is discussed. / Dissertation/Thesis / Ph.D. Biochemistry 2010

The p53 protein plays a central role in controlling the fate of cancer cells. At moderate levels of DNA damage, the concentration of the phosphorylated form of p53 undergoes temporal oscillation with a period of a few hours. In Dr. Shi’s lab, single-cell measurements were carried out using the p53-YFP fusion proteins and time-lapse fluorescence microscopy. We report here a detailed study of the image data. From the time series of the p53 concentration in individual cells, we deduce the amplitude and period of the oscillation. The pulse-to-pulse and cell-to-cell variability of the oscillation is characterized. We then carry out a computational study of a mathematical model that involves a negative feedback loop between p53 and Mdm2 proteins. We have determined the phase diagram of the model, and studied the sensitivity of the properties of the oscillating state against the model parameters. Although only p53 concentration is measured in the experiment, we show that careful analysis of the pulse shape can nevertheless yield valuable information on the underlying molecular processes, and shed light on the possible origin of the observed cellto- cell variations.

Master of Science / Department of Chemical Engineering / James Edgar / The bio-pharmaceutical industry began over 30 years ago with the production of human insulin and has shown incredible growth ever since. With forecasted annual worldwide sales of over $450B in 2025 for biopharmaceuticals, they are expected to be at least 25% of the entire pharmaceutical market. Therapeutics based on monoclonal antibodies (mAbs) make up roughly a third of all biopharmaceutical sales with indications from asthma, to cancer, to Parkinson’s disease. The recent approval of the first biosimilar mAb products in the US and Europe has exposed up to 20 of the top grossing biologic products to competition for the first time, while 75% of the US market is expected to lose patent exclusivity by 2020. With the increased competition from biosimilars, the costs associated with producing mAb based therapeutics will be a constraint on maintaining market share going forward. The majority of the total manufacturing costs for mAbs resides in the downstream processing where Protein A chromatography is the predominantly employed technology for the primary capture step. With Protein A’s high unit cost of up to $15,000 per liter and susceptibility to deamidification when exposed to high pH cleaning and sanitization chemicals, it is no surprise that many mAb manufacturers are considering alternatives. The objective of this work is to review the production process of mAb therapeutics, with a specific focus on the advantages, disadvantages, and alternatives to Protein A affinity chromatography as the primary capture step in downstream processing.

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