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Indolicidin is a broad-spectrum antimicrobial peptide 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 antimicrobial peptide 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 Ni2+-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 antimicrobial peptides for clinical and research applications.