Isolation, purification and activity identification of ABL tyrosine kinase and ABLT315I mutant / 中国生化药物杂志

ABSTRACT

Objective To establish a simple , stableand effective method for the isolation and purification of ABL tyrosine kinase and its mutant ABLT315I.Methods pET-28a vector was inserted in abl gene or its site directed mutagenesis.Then Escherichia coli BL21 competent cells were co-transformed with pGEX6P-1-ptp-1b and pET28a-abl/pET28a-ablc944t .The transformed BL21 cells were incubated, and then were stimulated with Isopropyl-β-D-thiogala-ctopyranoside ( IPTG ) to express ABL tyrosine kinase and its mutant .The ABL tyrosine kinase and its mutant was purified by affinity chromatography and gel filtration chromatography .SDS-PAGE was used to detect the purity and relative molecular weight of ABL tyrosine kinase and its mutant.BCA method was used to determine the concentration of ABL tyrosine kinase and its mutant .Finally, kinase activity of target protein was examined by ATP /NADH coupling method .ResuIts SDS-PAGE showed the high purity of ABL tyrosine kinase and its mutant.The concentration of ABL and ABLT 315I protein was reached 28mg/L of LB and 20mg/L of LB, respectively.Both of the target protein was measured to have good tyrosine kinase activity in vitro .ConcIusion A simple, stable and effective method for the isolation and purification of ABL tyrosine kinase and its mutant was found successfully in the study , which laying good foundation for High Throughput Drug Screening and structure analysis of protein subsequently .