CAJ | 학술논문

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pET－28a载体后，与pGEX6P－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。
목적：건립일충간단、은정、고효분리순화ABL락안산격매급기돌변체ABLT315I적방법。방법 abl급기정점돌변기인삽입pET－28a재체후，여pGEX6P－1－ptp－1b공동전화입대장간균BL21감수태세포중진행배양，가입이병기류대－β－D반유당감（IPTG）유도ABL락안산격매급기돌변체적표체，친화층석화응효과려층석순화단백，SDS－PAGE분석단백순도여상대분자량，BCA법측정단백농도，ATP／NADH우련법측정단백격매활성。결과 SDS－PAGE현시ABL급ABLT315I단백구유흔고적순도，병차기농도분별체도28 mg／L（ LB균액）화20 mg／L（ LB균액）。2충목적단백재체외균측득료흔호적락안산격매활성。결론본연구성공건립료일충은정、간단고효적ABL단백급기돌변체적분리순화방법，위후속단백진행고통량약물사선화결구분석건립량호적기출。
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 .