Publications (40) View all
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Article: High-Throughput Self-Interaction Chromatography: Applications in Protein Formulation Prediction
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ABSTRACT: PurposeDemonstrate the ability of an artificial neural network (ANN), trained on a formulation screen of measured second virial coefficients to predict protein self-interactions for untested formulation conditions. Materials and MethodsProtein self-interactions, quantified by the second virial coefficient, B 22, were measured by self-interaction chromatography (SIC). The B 22 values of lysozyme were measured for an incomplete factorial distribution of 81 formulation conditions of the screen components. The influence of screen parameters (pH, salt and additives) on B 22 value was modeled by training an ANN using B 22 value measurements. After training, the ANN was asked to predict the B 22 value for the complete factorial of parameters screened (12,636 conditions). Twenty of these predicted values (distributed throughout the range of predictions) were experimentally measured for comparison. ResultsThe ANN was able to predict lysozyme B 22 values with a significance of p < 0.0001 and RMSE of 2.6 × 10−4mol ml/g2. ConclusionsThe results indicate that an ANN trained on measured B 22 values for a small set of formulation conditions can accurately predict B 22 values for untested formulation conditions. As a measure of protein–protein interactions correlated with solubility, B 22 value predictions based on a small screen may enable rapid determination of high solubility formulations.Pharmaceutical Research 04/2012; 26(2):296-305. · 4.09 Impact Factor -
Article: Purification of CFTR for mass spectrometry analysis: identification of palmitoylation and other post-translational modifications.
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ABSTRACT: Post-translational modifications (PTMs) play a crucial role during biogenesis of many transmembrane proteins. Previously, it had not been possible to evaluate PTMs in cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial ion channel responsible for cystic fibrosis, because of difficulty obtaining sufficient amounts of purified protein. We recently used an inducible overexpression strategy to generate recombinant CFTR protein at levels suitable for purification and detailed analysis. Using liquid chromatography (LC) tandem and multiple reaction ion monitoring (MRM) mass spectrometry, we identified specific sites of PTMs, including palmitoylation, phosphorylation, methylation and possible ubiquitination. Many of these covalent CFTR modifications have not been described previously, but are likely to influence key and clinically important molecular processes including protein maturation, gating and the mechanisms underlying certain mutations associated with disease.Protein Engineering Design and Selection 11/2011; 25(1):7-14. · 2.94 Impact Factor -
Article: Hazard analysis and risk assessment in the development of biomedical drug formulation equipment.
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ABSTRACT: Hazard analysis and risk assessment techniques are utilized within many private sector industries and government agencies, including the medical device and pharmaceutical industry, within a structured process to control human injuries and environmental and property damage. In the U.S. the Federal Drug Administration (FDA) requires a hazard analysis be performed on all medical devices. While there are biomedical engineering applications reported which deal with human hazards in clinical, patient care environment, no previous studies extend these traditional techniques to a university-based, research environment. This study applies a tiered approach to hazard analysis and risk assessment to a biomedical, university-based, research environment in the design of a high throughput platform that screens chemical excipients (additives) for their ability to increase protein solubility. Each design stage (conceptual, preliminary, system, and detailed) requires a unique hazard analysis technique based on available information. The analysis techniques applied here are evaluated for their use in a biomedical research environment where experiment accuracy is a primary concern.Annals of biomedical engineering 11/2011; 40(4):898-906. · 2.41 Impact Factor -
Article: Self-interaction chromatography as a tool for optimizing conditions for membrane protein crystallization.
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ABSTRACT: The second virial coefficient, or B value, is a measurement of how well a protein interacts with itself in solution. These interactions can lead to protein crystallization or precipitation, depending on their strength, with a narrow range of B values (the 'crystallization slot') being known to promote crystallization. A convenient method of determining the B value is by self-interaction chromatography. This paper describes how the light-harvesting complex 1-reaction centre core complex from Allochromatium vinosum yielded single straight-edged crystals after iterative cycles of self-interaction chromatography and crystallization. This process allowed the rapid screening of small molecules and detergents as crystallization additives. Here, a description is given of how self-interaction chromatography has been utilized to improve the crystallization conditions of a membrane protein.Acta crystallographica. Section D, Biological crystallography 01/2010; 66(Pt 1):44-50. · 12.67 Impact Factor -
Article: Structure of nicotinic acid mononucleotide adenylyltransferase from Bacillus anthracis.
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ABSTRACT: Nicotinic acid mononucleotide adenylyltransferase (NaMNAT; EC 2.7.7.18) is the penultimate enzyme in the biosynthesis of NAD(+) and catalyzes the adenylation of nicotinic acid mononucleotide (NaMN) by ATP to form nicotinic acid adenine dinucleotide (NaAD). This enzyme is regarded as a suitable candidate for antibacterial drug development; as such, Bacillus anthracis NaMNAT (BA NaMNAT) was heterologously expressed in Escherichia coli for the purpose of inhibitor discovery and crystallography. The crystal structure of BA NaMNAT was determined by molecular replacement, revealing two dimers per asymmetric unit, and was refined to an R factor and R(free) of 0.228 and 0.263, respectively, at 2.3 A resolution. The structure is very similar to that of B. subtilis NaMNAT (BS NaMNAT), which is also a dimer, and another independently solved structure of BA NaMNAT recently released from the PDB along with two ligated forms. Comparison of these and other less related bacterial NaMNAT structures support the presence of considerable conformational heterogeneity and flexibility in three loops surrounding the substrate-binding area.Acta Crystallographica Section F Structural Biology and Crystallization Communications 11/2008; 64(Pt 10):893-8. · 0.51 Impact Factor
