ELP-Intein-Mediated Expression And Purification Of CA-MA Peptide

Elastin-like polypeptides (ELPs) are a class of syntheticmolecules with VPGXG repeats, which have a temperature-induced reversible inverse transition property. Inteins are a class of peptide segments that are spliced out from the precursor amino acid sequences in protein maturation process with an autocleavage property. In order to produce antimicrobial peptides efficiently, three different ELP-intein-mediated prokaryotic expression vectors were constructed in this study. By using the temperature-induced reversible inverse transition property of ELP and the autocleavage property of inteins, the expressed fusion proteins could be isolated easily by differential centrifugation at different temperatures. Then, the peptides of interest could be released from the fusuin tags in suitable solutions containing DTT. Therefore, the expression sytems can simplify the purification process and reduce the production costs of recombinant proteins, antimicrobial peptides in particular.Antimicrobial polypeptides (AMPs) have a wide variety of antibacterial, antiviral, antitumor and other biological activity. Hybrid CA-MA peptide is consisted of 1-8 amino acids of cecropin A (CA) and 1-12 amino acids of magaininⅡ(MA), which has been successfully expressed in both E.coli and yeast expression systems. To simplify the purification procedure and to reduce the cost of the AMP production, ELP andΔI-CM intein-containing expression vector pET-EIGFP was digested by restriction enzymes to remove the GFP reporter gene, resulting in ELP-intein-mediated expression vector pET-EI. Sce VMA intein sequence was amplified from vector pTYB11 by PCR and cloned into pET-EI vector after removal ofΔI-CM intein sequence by restriction digestion, resulting another expression vector pET-ESI. Ssp DnaB intein sequence was amplified from expression vector pTWIN1 by PCR and inserted into pET-EI vector after removal ofΔI-CM intein sequence by restriction digestion, resulting the third expression vector pET-MIE. The CA-MA coding sequence was amplified by overlapping PCR and cloned into each of the three vectors, resulting recombinant vectors pET-EICA-MA, pET-ESICA-MA and pET-MIECA-MA. The three recombinant vectors were transformed into E.coli and the fusion protein expression was induced at low-temperature gradient. The ELP-Intein-CAMA fusion proteins were purified by temperature shifting and differential centrifugation, and released from the protein tags by autocleavage. Tricine-SDS-PAGE showed that CA-MA peptide was expressed and purified from pET-EICA-MA transfromant with an expected molecular weight of 3 kDa. By using DH5αE. coli and Staphylococcus aureus as the indicator bacteria, antimicrobial activity of the CA-MA peptide was demonstrated by agar diffusion method and broth microdilution method with a minimal inhibitory concentration of 14.5μg/mL and 9.2μg/mL, respectively.