a novel pcr-based method for high throughput prokaryotic expression of antimicrobial peptide genes一种新型pcr进行高通量方法抗菌肽基因的原核表达.pdfVIP

Ke et al. BMC Biotechnology 2012, 12:10 /1472-6750/12/10 METHODOLOGY ARTICLE Open Access A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes 1,2*† 3† 3 2 1 2 2 2 Tao Ke , Su Liang , Jin Huang , Han Mao , Jibao Chen , Caihua Dong , Junyan Huang , Shengyi Liu , Jianxiong Kang4, Dongqi Liu4 and Xiangdong Ma3* Abstract Background: To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin- like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity. Results: Two or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against