TAILIEUCHUNG - Báo cáo khoa học: Using directed evolution to improve the solubility of the C-terminal domain of Escherichia coli aminopeptidase P Implications for metal binding and protein stability

There have been many approaches to solving problems associated with pro-tein solubility. This article describes the application of directed evolution to improving the solubility of the C-terminal metal-binding domain of amino-peptidase P fromEscherichia coli. | ễFEBS Journal Using directed evolution to improve the solubility of the C-terminal domain of Escherichia coli aminopeptidase P Implications for metal binding and protein stability Jian-Wei Liu1 Kieran S. Hadler2 Gerhard Schenk2 and David Ollis1 1 Research Schoolof Chemistry Australian National university Canberra Australia 2 Schoolof Molecular and MicrobialSciences University of Queensland Brisbane Australia Keywords directed evolution domain fusion metalloprotein protein solubility Correspondence . Liu Research Schoolof Chemistry Australian NationalUniversity Canberra ACT 2601 Australia Fax 61 2 6125 0750 Tel 61 2 6125 5061 E-mail jianw@ Received 10 May 2007 revised 4 July 2007 accepted 11 July 2007 doi 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

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