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Eur J Biochem. 2004 May;271(9):1623-9.

Phage-display as a tool for quantifying protein stability determinants.

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  • 1Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080, USA.


To address questions of protein stability, researchers have increasingly turned to combinatorial approaches that permit the rapid analysis of libraries of protein variants. Phage-display has proved to be a powerful tool for analyzing protein stability due to the large library size and the robustness of the phage particle to a variety of denaturing conditions. With the B1 domain of protein G (GB1) and a camelid heavy chain antibody as model systems, we are using phage-display libraries to experimentally address questions that have generally been addressed in silico, either through computational studies or statistical analysis of known protein structures. One effort has focused on identifying novel solutions to repacking the hydrophobic core of GB1, while maintaining stability comparable to the wild type protein. In a second study, a small set of substitutions in complimentarity-determining region 3 was found to stabilize the framework of the camelid antibody. Another major focus has been to obtain quantitative data on beta-sheet stability determinants. We have successfully adapted a phage-display method for quantitating affinities of protein variants (shotgun alanine scanning) to analysis of GB1 stability. Using this method, we have analyzed the energetic contributions of cross-strand side chain-side chain interactions. Finally, we discuss parameters to consider in using phage-display to discriminate subtle stability differences among fully folded variants. Overall, this method provides a fast approach for quantitatively addressing biophysical questions.

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