Title

A new strategy to produce active human Src from bacteria for biochemical study of its regulation

Document Type

Article

Date of Original Version

7-28-2006

Abstract

Enzymological studies of Src protein tyrosine kinase have been hindered by the lack of a suitable bacterial expression system. Poor expression of active Src appears to be due to toxicity associated with its kinase activity. To overcome this problem, we fused Src to a protein tyrosine phosphatase with an affinity tag and an appropriate thrombin cleavage site. Upon affinity purification of the fusion protein, Src was released by thrombin digestion and further purified by FPLC. This strategy has been used to produce several Src mutants that display catalytic and regulatory properties similar to those from eukaryotic expression systems. Characterization of the Src mutants confirmed that inactivation of Src by Csk through tail tyrosine phosphorylation required the Src SH3 domain. © 2006 Elsevier Inc. All rights reserved.

Publication Title

Biochemical and Biophysical Research Communications

Volume

346

Issue

2

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