Serial expression and activity analysis of LNK-16: a bovine antimicrobial peptide analogue.
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.
Department of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang, 453003, Henan, China.
Yanzhao
Y
Xu
Qing
Q
Wang
Bolin
B
Hang
Dengfeng
D
Fu
Tiantian
T
Shang
Zhiyu
Z
Zhao
Qinghua
Q
Zhang
Jian-He
JH
Hu
D000818:Animals; D000890:Anti-Infective Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D002176:Candida albicans / Q000187:drug effects; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli; D008826:Microbial Sensitivity Tests; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D011994:Recombinant Proteins; C073143:indolicidin