ABL酪氨酸激酶及突变体蛋白ABL^(T315I)的分离纯化及活性鉴定  

Isolation,purification and activity identification of ABL tyrosine kinase and ABL^(T315) Imutant

作　　者：刘素晓[1] 王幼平[1] 崔琳[1] 刘卫红[1] 沈思[1] 邢作英[1] 

机构地区：[1]河南中医学院第一附属医院中心实验室,河南郑州450000

出　　处：《中国生化药物杂志》2015年第8期19-22,共4页Chinese Journal of Biochemical Pharmaceutics

摘　　要：目的建立一种简单、稳定、高效分离纯化ABL酪氨酸激酶及其突变体ABLT315I的方法。方法 abl及其定点突变基因插入p ET-28a载体后,与p GEX6P-1-ptp-1b共同转化入大肠杆菌BL21感受态细胞中进行培养,加入异丙基硫代-β-D半乳糖苷(IPTG)诱导ABL酪氨酸激酶及其突变体的表达,亲和层析和凝胶过滤层析纯化蛋白,SDS-PAGE分析蛋白纯度与相对分子量,BCA法测定蛋白浓度,ATP/NADH偶联法测定蛋白激酶活性。结果 SDS-PAGE显示ABL及ABLT315I蛋白具有很高的纯度,并且其浓度分别达到28 mg/L(LB菌液)和20 mg/L(LB菌液)。2种目的蛋白在体外均测得了很好的酪氨酸激酶活性。结论本研究成功建立了一种稳定、简单高效的ABL蛋白及其突变体的分离纯化方法,为后续蛋白进行高通量药物筛选和结构分析建立良好的基础。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 .

关 键 词：慢性粒细胞白血病 ABL蛋白 酪氨酸激酶 药物筛选 

分 类 号：Q55[生物学—生物化学] Q51