Xu Yanzhao, Wang Qing, Hang Bolin, Fu Dengfeng, Shang Tiantian, Zhao Zhiyu, Zhang Qinghua, Hu Jian-He
Department of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang, 453003, Henan, China.
Protein J. 2014 Aug;33(4):309-12. doi: 10.1007/s10930-014-9563-0.
Indolicidin is a broad-spectrum antimicrobial peptide (AMP) with great therapeutic potential; however, high manufacturing costs associated with industrial-scale chemical synthesis have limited its delivery. Therefore, the use of recombinant DNA technology to produce this peptide is urgently needed. In this study, a new methodology for the large-scale production of a novel bovine AMP was developed. LNK-16 is an analogue of indolicidin that contains a kallikrein protease site at its C-terminus. The amino acid sequence of LNK-16 was synthesized using Escherichia coli-preferred codons. Three copies of the target gene were assembled in series by overlapping PCR and cloned into pET-30a(+) for the expression of His-(LNK-16)(3) in E. coli BL21 (DE3) cells. The expressed fusion protein His-(LNK-16)(3) was purified by Ni(2+)-chelating chromatography and then cleaved by kallikrein to release LNK-16. The recombinant LNK-16 peptide showed antimicrobial activity similar to that of chemically synthesized LNK-16 and indolicidin. Together, these data indicate that the use of serial expression can improve the large-scale production of AMPs for clinical and research applications.
吲哚杀菌素是一种具有巨大治疗潜力的广谱抗菌肽（AMP）；然而，与工业规模化学合成相关的高生产成本限制了其应用。因此，迫切需要使用重组DNA技术来生产这种肽。在本研究中，开发了一种大规模生产新型牛抗菌肽的新方法。LNK-16是吲哚杀菌素的类似物，在其C末端含有激肽释放酶蛋白酶位点。使用大肠杆菌偏好密码子合成了LNK-16的氨基酸序列。通过重叠PCR将三个目标基因串联组装，并克隆到pET-30a(+)中，以在大肠杆菌BL21(DE3)细胞中表达His-(LNK-16)3。表达的融合蛋白His-(LNK-16)3通过Ni2+螯合色谱法纯化，然后用激肽释放酶切割以释放LNK-16。重组LNK-16肽显示出与化学合成的LNK-16和吲哚杀菌素相似的抗菌活性。总之，这些数据表明，串联表达的方法可提高用于临床和研究应用的抗菌肽的大规模生产。
