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Using directed evolution to improve the solubility of the C-terminal domain of Escherichia coli aminopeptidase P Implications for metal binding and protein stability

Liu, Jian-Wei and Hadler, Kieran S. and Schenk, Gerhard and Ollis, David (2007) Using directed evolution to improve the solubility of the C-terminal domain of Escherichia coli aminopeptidase P Implications for metal binding and protein stability. The FEBS Journal, 274 (18). pp. 4742-4751. ISSN 1742-464X

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Abstract

There have been many approaches to solving problems associated with protein solubility. This article describes the application of directed evolution to improving the solubility of the C-terminal metal-binding domain of aminopeptidase P from Escherichia coli. During the course of experiments, the domain boundary and sequence were allowed to vary. It was found that extending the domain boundary resulted in aggregation with little improvement in solubility, whereas two changes to the sequence of the domain resulted in dramatic improvements in solubility. These latter changes occurred in the active site and abolished the ability of the protein to bind metals and hence catalyze its physiological reaction. The evidence presented here has led to the proposal that metals bind to the intact protein after it has folded and that the N-terminal domain is necessary to stabilize the structure of the protein so that it is capable of binding metals. The acid residues responsible for binding metals tend to repel one another ) in the absence of the N-terminal domain, the C-terminal domain does not fold properly and forms inclusion bodies. Evolution of the C-terminal domain has removed the destabilizing effects of the metal ligands, but in so doing it has reduced the capacity of the domain to bind metals. In this case, directed evolution has identified active site residues that destabilize the domain structure.

Item Type: Article
Keywords: directed evolution; domain; fusion; metalloprotein; protein solubility;
Subjects: Science & Engineering > Chemistry
Item ID: 3744
Depositing User: Gary Schenk
Date Deposited: 07 Jun 2012 14:59
Journal or Publication Title: The FEBS Journal
Publisher: Blackwell
Refereed: Yes
URI:

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