利用ELP-Intein系统在大肠杆菌中生产抗菌肽DLP4

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_N）和C端片段（I0_C）后，分别与ELP或DLP4融合，构建了pET-ELP-I0_N和pET-ELP-I0_C-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...

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

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_N) and C-intein (I0_C), and fused with ELP or DLP4 to construct two recombinant expression plasmids pET-ELP-I0_N and pET-ELP-I0_C-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.