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,G418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5 L罐培养,镍柱亲和纯化在250 mmol/L咪唑浓度下洗下PDGFRβ融合蛋白,Western blot验证约为90.08 kD,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-PDGFRp was constructed to express recombinant receptor tyrosine kinase PDGFR?and the right Pichia pastoris transformants were screened on to-deficient plates and YPD-G418 plates by turns after electroporation of strain GS115, a high yield strain named M3 was screened. The strain M3 was cultured in a 5 L fermentor and His-GFP-PDGFR?fusion protein was purified by Ni~(2+) 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 PDGFRP was successfully expressed in P. pastoris and could be used as a target for small molecule selective inhibitors screening.
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