Cloning, Expression and Purification of β-Defensins from S. salar (Atlantic Salmon)

Abstract

Introduction: The purpose of this study was to develop a method to recombinantly produce salmon β-defensins in the prokaryotic host Escherichia coli. β-defensins are a group of antimicrobial peptides (AMPs) which display potent and immunomodulatory properties in several species. Three expressed sequence tags (EST) corresponding to salmon β-defensins were previously identified by GenBank accession no: CK892029, CK895920 EG781611). It was hypothesised that these expressed sequence tags from the S. salar genome contain the genes corresponding to 3 β-defensin AMPs that are important mediators of the innate immune system of salmon. Given this information, the aim of this project is to clone these 3 genes into E. coli, followed by induction of overexpression of the gene products and purify these peptides using affinity chromatography. Methods: β-defensin genes 1, 3 and 4 were genes were commercially synthesised by Integrated DNA Technology and amplified using polymerase chain reaction (PCR). The β-defensin 4 gene was ligated into the pET44a expression plasmid creating a NusA-β-defensin 4 fusion gene. All 3 β-defensin genes were ligated into the pMAL-p4X expression plasmid creating MBP-β-defensin fusion genes. Only the MBP-β-defensin 4 fusion gene were transformed into Rosetta gami and BL21 (DE3) E. coli cells and expression trials were carried out using both IPTG (Isopropyl Β-D-1-Thiogalactopyranoside) and auto-induction for HIS tagged-NusA-β-defensin 4 fusion peptide and autoinduction was used to overexpress the MBP-β-defensin 4 fusion peptide. Nickel affinity chromatography using Nickel-NTA Sepharose resin was used to purify the resulting NusA-β-defensin 4 protein and amylose affinity resin was used to purify the MBP-β-defensin 4. Protein concentration and yield was quantified using both the Bradford and BCA assays and purity was assessed using sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and Western blotting. Results: β-defensins 1, 3 and 4 genes were successfully cloned and the sequences of the products were verified by Sanger sequencing. Expression studies were carried out with the β-defensin 4 peptide only. Optimal expression was successful using the pMALp4x expression plasmid in BL21 (DE3) cells with autoinduction for 48 hours at 37°C. A total of 5.2 mg of MBP-β-defensins 4 fusion protein were produced per litre of E. coli expression culture. xii Conclusion: The findings from this project have made a significant impact on the development of an in vitro laboratory method to produce recombinant Salmo salar β-defensins in E. coli. Although further work is needed to complete the cleavage of the fusion tag from the mature β-defensin, the majority of the protocol has been optimised and can be applied to β-defensin 1 and 3. The study has investigated important core strategies which central to future investigations of salmon β-defensins such as structural studies, minimum inhibitory concentration (MIC) assays and membrane permeability and amoebicidal assays.