摘要
刺桐胰蛋白酶抑制剂(ETI)是一种丝氨酸蛋白酶抑制剂,能够特异性结合胰蛋白酶及组织型纤溶酶原激活剂,抑制其活性。因此ETI可以作为配体与固定相偶联制备亲和层析介质,用于tPA(组织型纤溶酶原激活剂)及其突变体的纯化。利用基因工程方法构建了ETI的高表达工程菌株,诱导表达后目的蛋白占总蛋白的39.6%,经包涵体变性、复性、纯化,制备了纯度高于96%的ETI样品,收率达到了63.5%。利用纯化的ETI,偶联制备了亲和层析介质,偶联率达到99%。用制备的ETI亲和层析介质纯化瑞替普酶(r-PA),获得的r-PA样品纯度高于95%,生物学活性高于5×105U/mg。本研究为ETI的进一步生产及应用奠定了基础。
ETI is a kind of serine proteinase inhibitor, which inhibits both trypsin and tissue-type plasminogen activator. Immobilized ETI is an effective ligand for the affinity purification of these enzymes. High expression strain was constructed by genetic engineering, ETI sample with above 96% of purity was prepared, and the yield 63.5% could be obtained, 1 g CNBr-activated Sepharose 4B was conjugated to 42 mg ETI protein to get the affinity chromatography media and the conjugate rate was 99%, which was applied to purify the recombination of serine proteinase, such as r- PA, and the results appeared to be promising. The biological activity and the purity of the accepted r- PA was above 5 × 10 5 U/mg and 95% respectively. The results presented here lay a solid foundation for future application of ETI on pharmaceutical production.
出处
《药物生物技术》
CAS
CSCD
2010年第4期283-286,共4页
Pharmaceutical Biotechnology
