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, e.g., Journal
Biochemical and Biophysical Research Communications
Volume
346
Issue
2
Citation/Publisher Attribution
Wang, Yue Hao, Marina K. Ayrapetov, Xiaofeng Lin, and Gongqin Sun. "A new strategy to produce active human Src from bacteria for biochemical study of its regulation." Biochemical and Biophysical Research Communications 346, 2 (2006): 606-611. doi: 10.1016/j.bbrc.2006.05.180.