利用ELP-Intein系统在大肠杆菌中生产抗菌肽DLP4  

Production of antimicrobial peptide DLP4 in Escherichia coli using an ELP-Intein system

作　　者：姜宇 李秀 林瑛[1] JIANG Yu;LI Xiu;LIN Ying(College of Chemistry,Chemical Engineering and Biotechnology,Donghua University,Shanghai 201620,China)

机构地区：[1]东华大学化学化工与生物工程学院,上海201620

出　　处：《生物工程学报》2022年第6期2365-2376,共12页Chinese Journal of Biotechnology

基　　金：上海市自然科学基金(19ZR1471000);国家自然科学基金(31470836)。

摘　　要：DLP4 (defensin-like peptide 4)是一种新型昆虫防御素抗菌肽,对革兰氏阳性细菌具有强大的抗菌活性而且不易产生抗药性。本研究利用类弹性蛋白(elastin-like polypeptide, ELP)的相变特性和蛋白质内含子(intein, I)的C端断裂系统,通过构建重组表达质粒pET-ELP-I-DLP4,以大肠杆菌(Escherichia coli)作为宿主细胞,诱导表达后的重组蛋白通过简单的离心、pH和温度转变进行纯化得到DLP4。研究中发现,在表达纯化过程中蛋白质内含子发生了C端提前断裂。为了解决这一问题,将其断裂为N端片段(I0;)和C端片段(I0;)后,分别与ELP或DLP4融合,构建了pET-ELP-I0;和pET-ELP-I0;-DLP4两种重组表达质粒。分别在大肠杆菌中诱导表达,将表达后的菌液混合,使蛋白质内含子恢复C端断裂活性,最终得到的DLP4的得率约为1.49 mg/L。抑菌试验证明纯化的DLP4表现出预期活性,这为DLP4在原核系统中的表达纯化提供了有效途径。DLP4(defensin-like peptide 4) is a novel insect defensin, which has strong antibacterial activity against Gram-positive bacteria and is not susceptible to develop drug resistance. In this study, an elastin-like polypeptide(ELP) and an intein fusion system were used for production and purification of DLP4, which combined the characteristics of the phase transition of ELP and the C-cleavage of the intein. A recombinant expression plasmid pET-ELP-I-DLP4 was constructed and transformed into Escherichia coli. Subsequently, DLP4 was purified by simple centrifugation, alternation of pH and temperature. However, the C-cleavage of the intein occurred unexpectedly during the process of expression and purification. To solve this problem, the intein was split into N-intein(I0;) and C-intein(I0;), and fused with ELP or DLP4 to construct two recombinant expression plasmids pET-ELP-I0;and pET-ELP-I0;-DLP4, respectively. These two plasmids were transformed into E. coli separately. The mixture of the two cultures of E. coli strains restored the C-cleavage activity of the intein. This operation yielded DLP4 of about 1.49 mg/L. Antibacterial test confirmed that the purified DLP4 exhibited expected activity. Thus, this approach can be used as an effective way for DLP4 expression and purification in the prokaryotic system.

关 键 词：抗菌肽 ELP标签 蛋白质内含子 断裂蛋白质内含子 抑菌活性 

分 类 号：Q78[生物学—分子生物学] R914[医药卫生—药物化学]