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An efficient method for protein phosphorylation using the artificially introduced of cognate-binding modules into kinases and substrates

Paper ID Volume ID Publish Year Pages File Format Full-Text
25005 43551 2007 8 PDF Available
Title
An efficient method for protein phosphorylation using the artificially introduced of cognate-binding modules into kinases and substrates
Abstract

Protein phosphorylation is a major post-translational modification that regulates cellular signal transduction. The phosphorylation of substrate proteins by kinases requires cognate pairs of substrates and kinases. In addition, phosphorylation is mediated through both indirect and direct interaction between these kinases and substrates, which makes it difficult to effectively prepare large quantities of recombinant phosphorylated proteins. Here, we report a novel protein phosphorylation method involving the artificial introduction of cognate-binding modules into substrates and enzymes. This enhances the local concentration of substrates around enzymes so that the enzymatic reaction proceeds more efficiently. We prepared substrate proteins containing an SH3 domain at their N-terminus, and a kinase containing an SH3-binding motif at its C-terminus. This method was successfully applied to the phosphorylation of CrkII and the Vav DH domain, and we prepared 15N-labelled phosphorylated CrkII for NMR analysis.

Keywords
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresisSignal transduction; Phosphorylation; Domain engineering; Kinase; SH3
First Page Preview
An efficient method for protein phosphorylation using the artificially introduced of cognate-binding modules into kinases and substrates
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 131, Issue 4, 30 September 2007, Pages 458–465
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering