Prokaryotic expression and purification of human renin and mature renin / 中国生物制品学杂志

ABSTRACT

@#Objective To express and purify the human renin(REN)and mature renin(REN-340)in prokaryotic cells,and detect the concentration. Methods According to the human REN gene sequence(5972)registered in NCBI,REN and REN-340 gene fragments were artificially synthesized and cloned into vector p ET-28a(+)after E.coli codon optimization,and the recombinant expression plasmids 6 × His-REN-p ET and 6 × His-REN-340-p ET were constructed. The recombinant plasmids were transformed into competent E.coli BL21(DE3)and induced at different induction temperatures(20,25,30and 37 ℃)and different final concentrations(0. 1,0. 2,0. 4 and 0. 5 mmol/L)of IPTG. Recombinant REN and REN-340were induced under the optimal induction conditions,and the concentrations were detected by ELISA after nickel ion affinity chromatography with Ni-NTA. Results The recombinant expression plasmids 6 × His-REN-p ET and 6 × His-REN-340-p ET were constructed correctly as identified by restriction analysis and sequencing. The optimum induction temperature of recombinant REN and REN-340 was 37 ℃ and 20 ℃,and the best final concentration of IPTG was 0. 1 and 0. 2 mmol/L,respectively. The concentrations of purified recombinant REN and REN-340 were 9 148. 21 and 15 115. 31 m IU/m L,respectively. Conclusion The prepared recombinant REN and REN-340 proteins have high concentration with short production cycle,which are expected to be used as candidate quality control products and standard substances for external quality assessment(EQA).