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Physicochemical Factors Affecting Protein Aggregation: Biomolecular Engineering of Proteins for Enhanced StabilityHui Wang Unknown Date (has links)
Protein aggregation is commonly encountered during the manufacture of protein-based bioproducts in processing such as protein expression, purification, refolding, shipping and storage (Volkin and Middaugh, 1992; Brange, 2000). Aggregation may shorten the shelf-life of pharmaceutical proteins (Frokjaer and Otzen, 2005) and induce severe hypersensitivity (Rosenberg, 2006). In addition, several diseases ranging from Alzheimer’s disease to cystic fibrosis are associated with protein aggregation in the form of amyloid fibrils and plaques (Dobson, 1999; Luheshi et al., 2008). Hence, studies on protein aggregation, especially those dealing with high concentrations of proteins, are highly demanded in both academic and industrial laboratories. To address the aforementioned issues, physicochemical factors affecting protein aggregation were investigated systematically in this project. Strategies were developed to inhibit protein aggregation during renaturation and to enhance protein stability against aggregation during and after production, especially when dealing with high protein concentrations. ∆5-3-Ketosteroid isomerase (KSI) was used as a model for aggregation studies during protein renaturation due to its intrinsic aggregation properties. KSI was overexpressed as inclusion bodies (IBs) in Escherichia coli (E. coli). Cost- and time-efficient combination of chemical extraction and one-step affinity purification ensured the production of denatured KSI with high purity at high yield. Several key factors, including protein concentration and ionic strength, were determined to greatly influence KSI aggregation during renaturation. Polymer addition (PEG 3000 and Eudragit S-100) was found to alter KSI aggregation behaviour in a polymer-specific manner, as quantified using reversed phase-high performance liquid chromatography (RP-HPLC) analysis. Light scattering for second virial coefficient (SVC) measurement, surface plasmon resonance (SPR), and microfluidics were applied to study the fundamental mechanism of protein aggregation. Lysozyme was further introduced as a control protein for comparison with KSI. A rapid lumped method was established to measure specific refractive index (∂n/∂c) and SVC values for KSI and lysozyme, which provided quantitative and qualitative information on thermodynamic interactions of molecules in solution. SPR and microfluidics were also used to explore protein aggregation properties. To our best knowledge, it is the first time SPR and microfluidics have been used to investigate protein aggregation behaviour. Both SPR and microfluidics present significant potential for assessing protein aggregation and diagnosis or drug screening of protein aggregation related diseases. The chemical and physical stability of proteins needs to be maintained after successful refolding to ensure an acceptably long shelf life, especially at high protein concentration (Chang and Hermsdorf, 2002). The pharmaceutical effects of lectins on cell growth provided incentive for studies to improve their stability. Human galectin-2 (hGal-2, a homodimeric lectin) was used as a study model in this project. Mutations were introduced at one of the two Cys residues (C57A, C57M, and C57S). Only the C57M variant was highly expressed in bacteria in soluble form. No aggregate of this mutant was detected during 3 weeks of storage. hGal-2 C57M also facilitated site-directed introduction of poly(ethylene glycol) (PEG) into the remaining sulfhydryl group (Cys75). Product analysis revealed rather complete conjugation with one PEG chain per protein subunit in homodimer. Neither secondary structure alteration nor the absence of binding ability to a glycoprotein (asialofetuin) was observed. The results document the feasibility of tailoring a human galectin for enhanced stability against aggregation as well as monoPEGylation, which enables further testing of biological properties including functionality as a growth regulator and the serum clearance rate of hGal-2.
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Computational Studies of Enzymatic Enolization Reactions and Inhibitor Binding to a Malarial ProteaseFeierberg, Isabella January 2003 (has links)
Enolate formation by proton abstraction from an sp3-hybridized carbon atom situated next to a carbonyl or carboxylate group is an abundant process in nature. Since the corresponding nonenzymatic process in water is slow and unfavorable due to high intrinsic free energy barriers and high substrate pKa s, enzymes catalyzing such reaction steps must overcome both kinetic and thermodynamic obstacles. Computer simulations were used to study enolate formation catalyzed by glyoxalase I (GlxI) and 3-oxo-Δ5-steroid isomerase (KSI). The results, which reproduce experimental kinetic data, indicate that for both enzymes the free energy barrier reduction originates mainly from the balancing of substrate and catalytic base pKas. This was found to be accomplished primarily by electrostatic interactions. The results also suggest that the remaining barrier reduction can be explained by the lower reorganization energy in the preorganized enzyme compared to the solution reaction. Moreover, it seems that quantum effects, arising from zero-point vibrations and proton tunnelling, do not contribute significantly to the barrier reduction in GlxI. For KSI, the formation of a low-barrier hydrogen bond between the enzyme and the enolate, which is suggested to stabilize the enolate, was investigated and found unlikely. The low pKa of the catalytic base in the nonpolar active site of KSI may possibly be explained by the presence of a water molecule not detected by experiments. The hemoglobin-degrading aspartic proteases plasmepsinI and plasmepsin II from Plasmodium falciparum have emerged as putative drug targets against malaria. A series of C2- symmetric compounds with a 1,2-dihydroxyethylene scaffold were investigated for plasmepsin affinity, using computer simulations and enzyme inhibition assays. The calculations correctly predicted the stereochemical preferences of the scaffold and the effect of chemical modifications. Calculated absolute binding free energies reproduced experimental data well. As these inhibitors have down to subnanomolar inhibition constants of the plasmepsins and no measurable affinity to human cathepsin D, they constitute promising lead compounds for further drug development.
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