Tandem Expression Of Antimicrobial Peptides Protegrin-1 & PR-39 In E. Coli And Alfalfa

Antimicrobial peptide PR-39 is from swine, and has played a multifunction role in host innate immunity. PR-39 shows a broad spectrum of antimicrobial activity, and is chemotactic for neutrophils and capable of regulating vascular cell-cell interaction. It can inhibit invasion and metastasis of cancer cells and apoptosis of hypoxic endothelial cells. There is a significant interest in developing this peptide for pharmaceutical applications. Protegrin-1 (PG-1) was first discovered in porcine leukocytes. It kills microorganism by forming ion channels in cellular membranes. Due to the broad range of antimicrobial activity of PG-1, it is considered as a potential pharmaceutical agent too. There is no report about the heterologous expression of mature antimicrobial peptides PR-39 and PG-1. In this study, PR-39 and PG-1 were co-expressed in E. coli and alfalfa.1. Fusion expression of Protegrin-1, PR-39 gene from swine in Escherichia Coli To express swine antimicrobial peptide PG-1 and PR-39 in prokaryotic expression system, the part of codon of gene was replaced by codon preference of E. coli. Complementary primers were designed and the integrity of the PG-1 or PR-39 fragment was amplified by SOE-PCR. The amplified fragments were inserted into pGEX-4T-1, and the fusion expression plasmid pGEX4T-PG-1 and pGEX4T-PR-39 were constructed. The plasmids were transferred into E. coli BL21(DE3) and induced by IPTG SDS-PAGE and Western-blotting analysis showed that the recombinants had expressed the specific proteins. By Glutathione Sepharase 4B affinity chromatography, the fusion proteins GST-PG-1 and GST-PR-39 were obtained and purified. After cleaved by enterokinase, recombinant antimicrobial peptides, PG-1 and PR-39, were obtained. The yields of recombinant PG-1 and PR-39 were 1.38 mg/1 and 1.11 mg/1 respectively. The recombinant peptides showed antibacterial activities by growth inhibition method.2. Tandem expression of PR-39 and Protegrin-1 gene in Escherichia Coli To implement co-expression of antimicrobial peptide PR-39 and Protegrin-1 (PG-1) in prokaryotic expression system, a tandem gene fragment encoding PR-39 and PG-1 has been synthesized chemically. The cleavage site (Asn-Gly) of hydroxylamine hydrochloride was introduced between PR-39 and PG-1. The fragment was inserted into vector pGEX-4T-1 and expressed in Escherichia coli. The fusion protein GST-PR39-PG1, purified by affinity chromatography, was cleaved first by hydroxylamine hydrochloride to release recombinant PG-1 and then by enterokinase to release PR-39. Purification of recombinant PR-39 and PG-1 was achieved, and 1.5mg PG-1 and 1.9mg PR-39 were obtained respectively from 11 culture. The recombinant antimicrobial peptides showed antibacterial activity.3. Tandem expression of PR-39 and Protegrin-1 gene in Alfalfa To implement co-expression of antimicrobial peptide PR-39 and Protegrin-1 (PG-1) in alfalfa, a tandem gene fragment encoding PR-39 and PG-1, upstream of which the signal peptide geng was added, has been synthesized chemically. An intervening sequence (linker peptide) was introduced between PR-39 and PG-1. The fragment was inserted into plant express vector pC-1301-PMI which was constructed by our lab previously. At the same time, the plant express vectors containing single peptides were constructed. The express vectors plasmids were transformed into agrobacterium tumefaciens. Alfalfa was transformed by agrobacterium tumefaciens. After PCR detection,2 of alfalfa transfected with tandem genes,4 of alfalfa transfected with PR-39 and 5 of alfalfa transfected with PG-1 were obtained. The recombinant antimicrobial peptides showed antibacterial activity by growth inhibition method in vitro.