文献类型：期刊文献

中文题名：受体型酪氨酸激酶PDGFRβ在毕赤酵母中的表达与纯化

英文题名：Expression and Purification of Receptor Tyrosine Kinase PDGFRβ

作者：茆建胜[1];周祥山[1];张元兴[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2009

卷号：36

期号：10

起止页码：1460

中文期刊名：微生物学通报

外文期刊名：Microbiology China

收录：CSTPCD;;北大核心:【北大核心2008】；CSCD:【CSCD2011_2012】；

语种：中文

中文关键词：受体酪氨酸激酶;PDGFRβ;毕赤酵母;表达

外文关键词：Receptor tyrosine kinase, Pichia pastoris, Cloning, Expression, RTK activity

摘要：构建了受体酪氨酸激酶PDGFRβ的融合表达载体pPIC3.5K-PDGFRβ,转化毕赤酵母GS115,通过组氨酸营养缺陷型筛选,G4l8高拷贝菌株筛选,以及摇瓶诱导表达筛选,得到一株高表达PDGFRβ毕赤酵母菌株M3。对该菌株进行5L罐培养,镍柱亲和纯化在250mmol/L咪唑浓度下洗下PDGFRβ融合蛋白,Western blot验证约为90.08kD,酶联免疫反应检测表明融合表达的PDGFRβ具有高的酪氨酸激酶活性。为筛选其小分子抑制剂奠定了基础。
A fusion expression vector pPIC3.5K-PDGFRβ was constructed to express recombinant receptor tyrosine kinase PDGFRβ and the right Pichia pastoris transformants were screened on his-deficient plates and YPD-G418 plates by turns after electroporation of strain GSll5, a high yield strain named M3 was screened. The strain M3 was cultured in a 5 L fermentor and His-GFP-PDGFRβ fusion protein was purified by Ni2＋ chelating affinity chromatography. One distinct peak was obtained after elution with 250 mmol/L imidazole. Fusion protein was proved to be 90.08 kD by western blotting, and have tyrosine kinase activity by ELISA. Results showed that the receptor tyrosine kinase PDGFRβ was successfully expressed in P. pastoris and could be used as a target for small molecule selective inhibitors screening.