WANG Song,WANG Jun-ping,CHEN Mo,XU Yang,CHEN Fang,SHEN Ming-qiang,WANG Ai-ping,SU Yong-ping,KAI Li.Expression and identification of recombinant fusion protein of thrombopoietin mimetic peptide and growth hormone in Pichia pastoris[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(21):2037-2040.
WANG Song,WANG Jun-ping,CHEN Mo,XU Yang,CHEN Fang,SHEN Ming-qiang,WANG Ai-ping,SU Yong-ping,KAI Li.Expression and identification of recombinant fusion protein of thrombopoietin mimetic peptide and growth hormone in Pichia pastoris[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(21):2037-2040.DOI:
Expression and identification of recombinant fusion protein of thrombopoietin mimetic peptide and growth hormone in Pichia pastoris
并亚克隆至载体pPIC9K中,将所构建pPIC9K-rTMP-GH表达质粒转化酵母宿主细胞GS115,MD/MM平板及G418抗性筛选高表达株,然后进行试管补料分批表达, Western blot对表达产物进行鉴定,通过巨核细胞集落形成能力对其生物学活性进行初步检测。 结果 成功构建pPIC9K-rTMP-GH表达质粒,筛选到高表达rTMP-GH重组融合蛋白的巴氏毕赤酵母细胞株,初步证实所表达产物具有刺激巨核细胞集落形成的能力。 结论 成功实现了rTMP-GH融合蛋白在巴氏毕赤酵母中的高效表达。
Abstract
Objective To fulfill high-efficient expression of rTMP-GH
a recombinant fusion protein of thrombopoietin mimetic peptide and human growth hormone
in Pichia pastoris. Methods cDNA fragment coding for rTMP-GH was amplified by PCR
and subsequently cloned into the expression vector pPIC9K. The linearlized plasmid pPIC9K-rTMP-GH was transformed into Pichia pastoris GS115 cells and screened in MD/MM plates under pressure of G418. The recombinant fusion protein rTMP-GH was identified by Western blotting
and its biological activity was analyzed by its ability on colony formation of megakaryoblasts. Results The pPIC9K-rTMP-GH expression plasmid was successfully constructed. GS115 cells with high expression of rTMP-GH was screened out
and the rough purified rTMP-GH showed promotive effect on megakaryoblast colony formation. Conclusion High-efficient expression of rTMP-GH in Pichia pastoris is achieved.