重组人组织型纤溶酶原激活剂缺失变体的工业化制备与性质研究

将含有人组织型纤溶酶原激活剂缺失变体(K2tPA)重组表达质粒的工程菌经10L种子罐培养及100L发酵,IPTG诱导表达,其表达量为占菌体总蛋白的20％,表达产物经体外变复性、TI—Sepharose亲和层析、SP-Sepharose离子交换层析,每100L发酵液得重组人组织型纤溶酶原激活剂缺失变体(K2tPA)纯品4g,纯度达95％以上,比活大于500000IU／mg,内毒素及热源含量、宿主蛋白残留量、宿主DNA残留量等均达到临床使用标准．其分子量(质谱测定)、氨基酸组成、N、C-端氨基酸序列分析等均与理论值相符．同时进行了等电点测定及肽图分析等性质研究．

Large Scale Preparation and Characterization of Recombinant Human K2tPA

The engineered E.coli containing an expression vector for recombinant human K_2tPA was cultured in a 10 L seeding tank, and then grew in a 100 L fermentor by induction of IPTG. The expressed protein accounted for 20% of the total bacteria proteins. After renaturation, the folding solution was applied to an affinity column of TI Sepharose, and the fraction containing K_2tPA activity was further purified by SP Sepharose ion exchange chromatography and depynogened by affi prep polymyxin affinity chromatography. A pilot purification yielded 4 g of purified K_2tPA from 100 L medium. The purity of the resulting protein was higher than 95%, and its specific activity was over 500 000 IU/mg. The trace content of pyrogen, the residual protein and DNA from the host cell all met the requirements for clinical use. The molecular weight weasured by mass spectrum, the amino acid composition and the N/C terminal analysis of K_2tPA were consistent with the theoretical data. The isoelectrophoretic point and peptide mapping of K_2tPA were also determined.