Bcr-Abl酪氨酸蛋白激酶域及其突变体的构建和表达

目的:制备Bcr-Abl激酶域及其突变体-His的重组蛋白. 方法:用PCR方法从含有Bcr-Abl全长的质粒pGD210中扩增出Bcr-Abl激酶域片段,再通过重组PCR技术获得激酶域的几个主要突变体(T315I,Y253F,E255K,E255V),测序正确后将其克隆到原核表达载体pET-32a中,构建表达激酶域片段和His标签融合表达的载体,IPTG诱导表达,得到的融合蛋白用镍-次氮基三乙酸(Ni-NTA)琼脂糖进行亲和层析纯化. 结果:成功得到了Bcr-Abl激酶域及其突变体序列,经序列分析证实后将重组质粒转入BL-21进行表达. 结论: 通过重组PCR技术获得了野生型激酶域及其突变体蛋白. 融合蛋白表达在包涵体,占菌体总蛋白的70%以上. 经Ni-NTA镍珠纯化后,纯度在95%以上.

Production, expression and purification of Bcr-Abl tyrosine kinase domain and its mutants

AIM: To prepare the Bcr-Abl protein tyrosine kinase domain and its mutants,which will lay a foundation for the study on the resistance to imatinib.METHODS: PCR was used to (amplify) the sequence of Bcr-Abl protein tyrosine kinase domain from plasmid pGD210.Its main mutants(T315I,Y253F,E255K,E255V) were got by recombinant PCR and cloned into prokaryotic expression vector pET-32a after sequencing,and the recombinant plasmid was expressed in E.coli strain BL21 induced by IPTG.The fusion protein was purified from the cell lysates by Ni-NTA affinity chromatography and analyzed by SDS-PAGE.RESULTS: The sequence of Bcr-Abl protein tyrosine kinase(domain) was successfully cloned, and was identical to that in GenBank.Point-mutants were got by recombinant PCR and then the recombinant plasmid was expressed in BL-21.CONCLUSION: The fragments of Bcr-Abl kinase domain and its mutants can be expressed in inclusion bodies,accounting for over 70% of total bacterial proteins.And the fusion protein can be purified to over 95% using Ni-NTA affinity chromatography.It will facilitate the research of chronic myeloid leukemia treatment.