纤维连接蛋白C端肝素结合域多肽在毕赤酵母中的表达、纯化及鉴定

Expression,purification and identification for fibronectin C-terminal heparin-binding domain polypeptide in Pichia pastoris

为在毕赤酵母中表达纤维连接蛋白C端肝素结合域(Fibronectin C-terminal heparin-binding domainFNCHBD)多肽并研究其功能,通过PCR技术扩增FNCHBD目的基因,将目的基因与T载体连接,经测序正确后,插入pAo815SM酵母表达载体增加基因拷贝数,然后酶切克隆入酵母表达载pPIC9K;将重组质粒Sal I酶切线性化后转化毕赤酵母菌株,筛选工程菌,经甲醇诱导表达,用SDS-PAGE检测发酵上清液,表明有重组蛋白FNCHBD多肽的高表达,表达产物通过离心、超滤、离子交换层析纯化,纯化产物通过SDS-PAGE、Western blotting印迹、质谱及肝素亲和层沉析对表达产物进行鉴定。结果表明利用酵母工程菌成功表达和纯化了FNCHBD多肽,多肽的分子量接近32 kDa,纯化产物的纯度可达95%以上,能被FN多克隆抗体特异识别且具有多肽肝素结合活性,为后续结构及功能的研究奠定基础。

To express and identify fibronectin C-terminal heparin-binding domain （FNCH BD） polypeptides in Pichia pastoris expression system and study its function, the fragment of FNCHBD was amplified by PCR and inserted into pGEM-T vector. After sequenced, the fragment was inserted into pAo815SM vector, and then cloned into the expression vector pPIC9k. The recombinant plasmid was linerarized with restrict enzyme Sal I and transferred into the yeast host cell KM71 and GS115. The positive yeast clone was screened by G418 resistant, and the target protein was induced to express in the medium containing 0.5% methano1. The culture supernatant was collected and then was purified with membrane ultrafiltration and ion exchange chromatography. The purified product was analyzed with mass spectrogram, SDS-PAGE, Western blotting and heparin affinity chromatography. The results showed that the target protein was around 32 kDa and the purity of the product was above 95%. FNCHBD could be specifically recognized by fibronectin polyclonal antibody. These results suggest that FNCHBD could be expressed and purified successfully in Pichia pastoris, which provides a good strategy to further studies.