重组TFPI缺失突变体TFPI1-161在毕赤酵母中的高效表达及功能鉴定

人组织因子途径抑制物(TFPI)是一种体内天然存在的外源性凝血途径特异性抑制物。缺失突变体TFPI1-161包括TFPI的N末端、K1和K2结构域，是一种研究TFPi结构与功能及其相互关系的理想对照分子。以克隆质粒pGEM-3Zf(-)-TFPi为模板，用PCR方法获得TFPI1-161基因，构建表达质粒pPIC9K-TFPi1-161并转化毕赤酵母GS115。通过筛选多拷贝转化子及优化发酵培养条件，首次在毕赤酵母中高效表达了TFPI1-161经纯化后最终产量高于酿酒酵母20倍以上。由于糖基化程度不同，TFPI1-161表达为TFPI1-161(24kD)和TFPI1-161(27kD)两种分子形式，其等电点分别为4、8和4．9。根据等电点差异，二可通过阴离子交换层析得到分离，其活性无显性差异。经分子筛和阴离子交换层析分离纯化后，从4L发酵培养液中可分别获得1.4g TFPI1-161(24kD)和1.8gTFPI1-161(27kD)，其比活性分别达12880u／mg和12400u／mg，回收率达55％。经稀释的凝血酶原时间及发色底物法检测，重组TFPI1-161具有良好的抗凝及抑制FXa活性的作用。为获得大量TFPI1-161提供了一种廉价高效的蛋白表达纯化方式，为进一步的基础及临床前研究奠定了基础。

High Expression and Characterization of Recombinant Deletion Variant TFPI1 - 161 of Human Tissue Factor Pathway Inhibitor in Pichia pastoris

Human tissue factor pathway inhibitor (TFPI) is a native specific inhibitor of extrinsic pathway of coagulation in vivo. As a kind of deletion variant which only contains N-terminus, K1 and K2 of TFPI, TFPI 1-161 is an ideal control molecule in the research of structure and function of TFPI. In this article, we not only investigated the methods of expression and purification, but also detected characteristics of TFPI 1-161. Using plasmid pGEM-3Zf(-)-TFPI as a template, DNA of TFPI 1-161 was obtained by PCR and cloned into plasmid pPIC9K to construct expression plasmid pPIC9K-TFPI 1-161. By force of screening multi-copy transformant and optimizing ferment condition, TFPI 1-161 was highly expressed in Pichia pastoris GS115 for the first time and the final yield after purification was increased more than 20-fold comparing with Saccharomyces cerevisiae. TFPI 1-161 was expressed as two different molecular weight proteins namely TFPI 1-161(24kD) and TFPI 1-161(27kD) because of difference of glycosylation. Their isoelectric points were 4.8 and 4.9 respectively. They were separated according to different isoelectric points using anion-exchange chromatography and had similar activity. Approximately 1.4g TFPI 1-161(24kD) and 1.8g TFPI 1-161(27kD) was obtained from 4-liter fermentation culture after purification by ultrafiltration, gel filtration, anion-exchange chromatography. Their specific activity were up to 12 880u/mg and 12 400u/mg respectively. The rate of recovery of TFPI 1-161 was about 55%. The recombinant TFPI 1-161 had good anticoagulant activity in diluted prothrombin time assay (dPT) and anti-FXa activity in a direct amidolytic assay. The expression and purification system described here provides a cost-effective method for generating large amounts of material for further fundamental and preclinical research of TFPI 1-161.