重组鼠源性肠激酶轻链在大肠杆菌中的表达

Expression of recombinant mouse enterokinase light chain in E.coli

目的:制备能在原核表达系统中表达并特异性识别(Asp)4-Lys序列的鼠源性肠激酶,为推广应用重组肠激酶提供技术平台。方法:采用RT-PCR从C57BL/6J小鼠的十二指肠肠系膜黏膜组织中钓取肠激酶轻链的cDNA,将其克隆入pET32a原核表达载体中,并在大肠杆菌BL21(DE3)中进行表达,然后以镍亲和层析法对表达产物进行纯化。结果:所钓取的肠激酶轻链编码序列与GenBank中的序列一致,比较发现其编码的氨基酸序列在小鼠与人、牛之间的同源性大于75%。利用pET32a/BL21(DE3)表达系统成功地在大肠杆菌中表达了重组鼠源性肠激酶轻链,表达量约占大肠杆菌BL21(DE3)总蛋白的30%,但多以包涵体的形式存在。经镍亲和层析法纯化的重组肠激酶多以聚体形式存在。结论:鼠源性肠激酶轻链在原核细胞中多以包涵体形式表达,天然构象重组肠激酶的获得还需对表达系统进行优化。

Objective To establish a prokaryotic expression system for expressing mouse enterokinase which could recognize and cut the sequence of （Asp）4-Lys in order to provide a technique platform for application of recombinant enterokinase. Methods The cDNA coding sequencing of enterokinase light chain was amplified by RT-PCR from mouse dodecadactylon mucosa and cloned into pET32a expression plasmid. The recombinant enterokinase light chain was expressed in BL21 （DE3） and purified with Ni-affinity chromatography. Results The cDNA sequence amplified was identical with that in GenBank and possessed more than 75% homologous in amino acid sequence with that from human and cow. The recombinant mouse enterokinase light chain could be well expressed in E. coli, but most of them were inclusion. Almost all recombinant enterokinase proteins were polymers after purification by Niaffinity chromatography. Conclusion Recombinant mouse enterokinase can be expressed in E. coli in inclusion manner so that the prokaryotic expression system for recombinant enterokinase with native conformation needs to be optimized.