重组羧肽酶B在胰岛素原C肽制备工艺中的应用

Application of Recombinant Carboxypeptidase B in the Preparation of Proinsulin C-peptide

用RT-PCR方法克隆了大鼠羧肽酶原B的cDNA编码序列,将其重组到原核表达质粒pT7-473中,并在大肠杆菌中以包涵体方式获得高表达。SDS-PAGE电泳及灰度扫描显示表达量达菌体总蛋白的35%,表达产物融合了表达质粒上的6His。在变性条件下,经Ni-NTA柱纯化,得到羧肽酶原B,在复性液中进行稀释复性后,胰蛋白酶切得具酶活性的羧肽酶B,然后经DEAE-FF分离纯化获得较纯的羧肽酶B(28.5mg/L),比活为13.5u/mg。用RP-HPLC分析胰蛋白酶+羧肽酶B酶解胰岛素原C肽三聚体的酶解产物,证明该重组的羧肽酶B可替代提取的羧肽酶B应用于胰岛素原C肽的制备工艺中。

The coding sequence of procarboxypeptidase B (proCPB) gene was obtained by RT-PCR from a fresh rat pancreas, subsequently cloned into the expression vector pT7-473 and transformed into (E.coli) BL21(DE3). After induction with (1 mmol/L) isopropyl β-D-thiopalactoside, the recombinant procarboxypeptidase B was highly expressed as inclusion bodies. SDS-PAGE analysis revealed that recombinant procarboxypeptidase B accumulated up to 35% of the total proteins of E.coli. The expression product was purified with Ni-NTA affinity column under denature condition after resolving the inclusion body with (8 mol/L) urea. The recovered procarboxypeptidase B being treated under folding conditions, the resulting folded procarboxypeptidase B was subjected to enzymatic cleavage to produce enzymatically active carboxypeptidase B, which was purified subsequently with DEAE-FF column to get the active carboxypeptidase B with special activity of about (13.5 u/mg). It was confirmed that recombinant carboxypeptidase B had the same catalytic activity as standard carboxypeptidase B purified from porcine pancreas through (digesting) the fusion multiple polymers proinsulin C-peptide.