Role of ELP_(30)-intein-tag in prokaryotic expression of recombinant lipoprotein-associated phospholipase A_2
10.13200/j.cnki.cjb.003989
- VernacularTitle:ELP_(30)-intein标签在重组脂蛋白相关磷脂酶A_2原核表达中的作用
- Author:
RUAN Yao
- Publication Type:Journal Article
- From:
Chinese Journal of Biologicals
2023;36(9):1080-1084+1096
- CountryChina
- Language:Chinese
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Abstract:
Objective To investigate the role of ELP_(30)-intein-tag in the prokaryotic expression of recombinant lipoproteinassociated phospholipase A_2 (LP-PL A_2 ). Methods The recombinant plasmid pIG6-ELP_(30)-intein-LP-PL A_2 was transformed into E.coli W3110. Using the recombinant protein LP-PL A_2 without ELP_(30)-intein-tag as negative control,the expression of recombinant ELP_(30)-intein-LP-PL A_2 in periplasm under different temperatures(20,25 and 30 ℃)and induction methods(IPTG and automatic induction)was analyzed by Western blot. The target protein was purified and isolated via inverse transition cycling(ITC)based on elastin-like polypeptide(ELP)-tag,and then detected by SDS-PAGE and Western blot.Results The recombinant LP-PL A_2 protein without ELP_(30)-intein-tag was expressed only intracellularly and not located in the periplasmic space. Under the same induction temperature,the amount of target protein induced by automatic induction was much lower than that induced by IPTG. Compared with 20 ℃,the overall expression level of target protein induced by IPTG increased at 25 ℃ and 30 ℃. The target protein ELP_(30)-intein-LP-PL A_2 was successfully isolated and purified by ITC technique,and the recombinant protein LP-PL A_2 was obtained with the purity of about 70% by inducing intein self-cleavage,which showed specific binding to the anti-LP-PL A_2 antibody. Conclusion The ELP_(30)-intein-tag can promote the expression of recombinant protein LP-PL A_2 in the periplasmic space of E.coli,and the recombinant protein LP-PL A_2 isolated by non-chromatographic purification has high purity,which provides a new method for the rapid,low-cost and effective production of recombinant protein LP-PL A_2 .
关键词(KeyWords)： 脂蛋白相关磷脂酶A_2;类弹性蛋白;原核表达
Lipoprotein-associated phospholipase A_2(LP-PL A_2);Elastin-like polypeptide(ELP);Prokaryotic expression
