Expression, Purification And Antimicrobial Activity Of Anti-staphylococcal Chimeric Protein P128 Using SPM-ELP Fusion Tag

Antibacterial peptides (ABP) are a class of small polypeptides with anti-bacterial, anti-viral, anti-fungal and/or anti-tumor activities in humans, animals and plants. Due to their unease of forming antibiotic-resistance, ABPs are becoming hot research topics in medicine, agriculture and biotechnology. Among these, P128-is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. The P128 protein has been expressed successfully in E. coli and purified by two-step of affinity chromatography, which is complex costly process.Elastin-like polypeptides (ELP) are a class of pentopeptides with temperature-sensitive reverse phase transition property, and therefore are present as a soluble form in solutions below their transition temperature (Tt) and become aggregates in the solutions above the Tt. ELP fusion proteins remain the reverse transition property of ELP and thus can be purified from recombinant bacterial extracts simply by centrifugation and/or filtration at suitable temperature and salt concentration. The 250-residue-long self-processing module (SPM) of the Neisseria meningitidis FrpC protein has the self-cleavage property and has been used as the C-terminal affinity tag for recombinant protein purification. A simple, rapid and economical recombinant protein expression and purification system has been established and used successfully to express and purify anti-bacterial peptides such as human defensin β. In this study, we applied this system to express and purify recombinant P128.The coding sequence for P128 was adapted to E. coli codon usage using JAVA Codon Adaption Tool and chemically synthesized sequence was cloned into SPM-ELP fusion expression vector pET-SPM-ELP. The construct was transformed into BL21 (DE3) E. coli and the expression of fusion protein was induced with IPTG SDS-PAGE analysis showed that the fusion protein was efficiently expressed in the recombinant E. coli as a soluble protein. After systematic optimization the parameters for transition cycling such as transition temperature, NaCl concentration and centrifugation force, the fusion protein was purified to a single protein band with a recovery rate of 35.4% by two rounds of ITC under the optimized conditions. The self-cleavage of fusion protein was induced overnight 25 ℃ with 20μM calcium and the released P128 was recovered by additional round of ITC with a purity of 89% and a yield of 960mg/L. Both agar plate diffusion and minimum inhibition concentration assays showed that the purified recombinant P128 had a potent anti-bacterial activity against Staphylococcus aureus with a minimum inhibition concentration of 8.4μg/ml.