人组织因子途径抑制因子在毕赤酵母中的表达与纯化

Expression and Purification of Tissue Factor Pathway Inhibitor in Pichia Pastoris

目的利用毕赤酵母高效表达人组织因子途径抑制因子(TFPI)。方法利用基因定点突变技术对人TFPIcDNA特定序列进行定点突变(同义突变)。将获得的突变体及野生型cDNA分别插入表达载体pPic9中,转化酵母宿主菌GS115和KM71,用0.5%的甲醇诱导重组蛋白表达。细胞培养上清经超滤浓缩后,依次用DEAE-sepharose Fast Flow、Heparin-sepharose CL6B及Sephadex G-75柱层析分离纯化得到重组蛋白,分别采用凝胶扫描成像定量分析和底物显色法测定重组蛋白的表达量和活性。结果凝胶扫描成像定量分析显示,同义突变体的表达量(1mg/L)显著高于天然型(0.1mg/L)。活性测定表明GS115重组菌在诱导表达12h后即可于培养上清中检测到TFPI活性,36h达峰值;而KM71重组菌诱导表达24h后才开始于培养上清中检测到TFPI活性,72h达峰值。两个菌株中突变体mTFPI-pPic9转化子的表达量均显著高于TFPI-pPic9转化子。重组蛋白相对分子质量约为42000。结论对TFPI-cDNA特定序列进行同义突变后能显著提高重组蛋白在毕赤酵母细胞中的表达,且显著高于在酿酒酵母和昆虫细胞的表达;重组蛋白具有良好的生物活性。因此,利用毕赤酵母表达TFPI是一种较好的选择。

Objective To generate recombinant human tissue factor pathway inhibitor （TFPI） in Pichia pastoris. Methods To improve the expression of TFPI, a silent mutation was generated at the specific site of TFPI cDNA. Both wild-type TFPI cDNA and mutated TFPI cDNA were cloned into the expression vector pPic9. The constructed plasmids were subsequently transformed into Pichia pastoris cells GS115 and KM71, and the transformants were confirmed by polymerase chain reaction and DNA sequencing. The expression of recombinant protein was induced by addition of 0. 5% methanol in the culture medium. The cell culture medium after induction was concentrated through ultra filtration. The recombinant protein was further purified by a three-step process （Heparin-sepharose CL-6B affinity chromatography, DEAE-Sepharose Fast Flow affinity chromatography, and Sephadex G75-gel filtration）. The amount of the recombinant protein was quantified with gel imaging system. The activity of the recombinant protein was analyzed by the chromogenic substrate assay. Results The amount of TFPI expressed in the mutated clone （ 1 mg/L） was much higher than that in the wild type clone （0.1 mg/L）. The TFPI activity in the recombinant GS115 cells could be detected 12 hours after induction and reached the peak at 36 hours, while the TFPI activity in the recombinant KM71 cells started to show up at 24 hours after induction and reached the peak at 72 hours. The expression of recombinant protein in the silent mutant was significantly higher than those of wild type clone in both GS115 and KM71 host cells. The relative molecular mass of recombinant TFPI was approximately 42 300. Conclusion Introduction of the silent mutation at the specific site of TFPI cDNA can increase the recombinant protein expression in Pichia pastoris, which is much higher than that in insect cells or saccharomyces cerevisiae.