A new strategy to produce active human Src from bacteria for biochemical study of its regulation.
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.
Wang
Yue-Hao
YH
Department of Cell and Molecular Biology, University of Rhode Island, Kingston, 02881, USA.
Ayrapetov
Marina K
MK
Lin
Xiaofeng
X
Sun
Gongqin
G
D001419:Bacteria / Q000201:enzymology*; D002352:Carrier Proteins / Q000235:genetics; D004789:Enzyme Activation; D006801:Humans; D058619:Maltose-Binding Proteins; D009154:Mutation; D010766:Phosphorylation; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013917:Thrombin / Q000378:metabolism; D019061:src-Family Kinases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D019061:src-Family Kinases; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D013917:Thrombin