The Genetic Recombinant Expression Of Antimicrobial Peptides Of Styela And Human

Defensins, a class of small cationic peptides, have been discovered to have broad anti-microbial activities and function as host defense peptides. The defensins have little toxic side effects and low risks of drug-resistance due to its unique anti-microbial mechanisms compared to widely-utilized antibiotics.The defensins have been currently found in various types of organisms. For instances the clavanins are a family of four a-helical antimicrobial peptides that were purified from the invertebrate Styela clava, a solitary tunicate. Clav E is a more potent member of this family shows to be less affected by pH values but with different characters of anti-microbial mechanisms in varied pH environments. Whereas the human defensins function in a pH-dependent manner in most cases and might lose their functions in specific pH enviroments.In this study we aimed at the recombinant expression of ascidian antimicrobial peptides ClavE and human defensins HD-5, HBD-2, and HBD-3 via genetic engineering methods. In order to combine the benefits of these 2 types of antimicrobial peptides that the chimera expression of ClavE with HD-5, HBD-2, and HBD-3 were respectively carried out by recombination as well. The coding genes of these anti-microbial peptides and fusion proteins were synthesised and recombined into prokaryotic and/or eukaryotic plasmid vectors and expressed in colibacillus and/or yeast. With the analyses of the microbial-inhibition activities and ranges of the recombinant proteins that we expected to obtain recombinant anti-microbial peptides with higher stability, broader pH tolerance range, and stronger microbial inhibitory ability.The coding DNA sequences of ClavE, HD-5, HBD-2, and HBD-3 were synthesized and recombined by exon optimization and overlap PCR. Thus the coding DNA sequences of ClavE-HD-5, ClavE-HBD-2, and ClavE-HBD-3 were then synthesized. All these coding genes were used to construct the following vectors:a) 7 eukaryotic vectors:pVT102U-a-HD5, pVT102U-a-HBD2, pVT102U-a-HBD3, pVT102U-α-ClavE, pVT102U-α-ClavE-HD5, pVT102U-α-ClavE-HBD2, pVT102U-α-ClavE-HBD3; b) 2 prokaryotic vectors:pET30a-ClavE-HD5, pET30a-ClavE-HBD3. Both eukaryotic and prokaryotic plasmids were then transformed into saccharomyces cerevisiae S78 and E coli. Rosetta (DE3) respectively. The positively transformed cells were used to express the target proteins.The expression products were analysed by SDS-PAGE gel which showed the bands at the corresponding melocular weight positions. It illustrated the successful expression of the target polypeptides. The anti-microbial effects of the expression products were tested by using the bacteriostatic ring method. The results indicate the successful expression of ClavE, HD-5, HBD-2, and HBD-3 in eukaryotic S. cerevisiae expressing system. The recombinant culture supernatant HD-5, HBD-2, and HBD-3 was proved to have anti-microbial activities by the anti-microbial test respectively. However, the production yield of these proteins seemed relatively low. The chimera protein of ClavE-HBD-3 and ClavE-HD5 were expressed in the prokaryotic Rosetta (DE3) strain. However both of the fusion proteins were in the inclusion body according to the solubility analysis.In this study, the expression of various antimicrobial peptide genes and their recombination in eukaryotic and/or prokaryotic expressing systems are discussed. It provides an essential fundation for later research on the activities of natural and fused defensins.