摘要
将基因工程菌株E .coliBL2 1(DE3)pET2 2b mETIa高密度发酵 ,用异丙基硫代 β D 半乳糖苷 (IPTG)诱导 ,重组刺桐胰蛋白酶抑制剂a (rETIa)蛋白在E .coli中得到较高水平表达 ,表达量占菌体总蛋白的 4 0 %以上 .经菌体破碎、包涵体变性、复性 ,二步柱层析纯化得到电泳纯的rETIa蛋白 .测得rETIa对t PA突变体(NTA)的抑制平衡常数Ki 为 8 72× 10 -8mol/L .据此利用纯化的rETIa蛋白制备rETIa Sepharose 4B亲和层析柱 .直接一步纯化NTA复性液 ,纯化的NTA纯度达 90 %以上 ,收率为 96 2 % ,纯化倍数为 13 2 ,比活为(5 6 5 7± 71 3)U/μg .
Being cultured in high cell density, E. coli BL21 (DE3) harboring plasmid PE722b-mETIa were induced by IPTG and then recombinant ETIa were highly expressed. Expressed rETIa were above 40% of total bacterial protein. After primary purification through breaking E. coli, dissolving inclusion bodies, refolding, and further purification by two-step chromatographies, rETIa of electrophoretic purtity has been obtained. Inhibitory activity of rETIa against tPA deletion variant (NTA) has been detected and inhibitory constant (K-i) was 8.72 x 10(-8) mol/L. So affinity chromatography column of rETIa-Sepharose 4B was prepared for purification of NTA. After only one-step purification with this column from refolded NTA, 13.2-fold puritied NTA with the specific activity of (565.7 +/- 71.3) U/mug and above 90% of purity, have been obtained with the recovery rate of 96.2%.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2002年第3期420-423,共4页
Progress In Biochemistry and Biophysics
