Expression,Purification And Antibacterial Activity Of Anti-staphylococcal Protein P128 Against Bovine-derived Staphylococci

P128 is the hybrid peptide consisting of the degrading enzyme active region of staphylococcal phage K cell wall and the cell wall binding domain of lysostaphin,with strong anti-staphylococcal activity.Recombinant P128 has been successfully expressed in Escherichia coli(E.coli),but its purification requires two-step precipitation and affinity chromatography.In our previous study,P128 was expressed as an elastin-like polypeptide(ELP)fusion protein,and the ELP tag was removed by self-processing module(SPM),but the cleavage efficiency was low and not reproducible.Tobacco etch virus(TEV)protease is the catalytic core region of the viral inclusion complex protease with the advantages of highly specific activity,less influence by amino acid sequences of target proteins,broad compatibility in different buffers.In our previous study,by fusing with a self-aggregating peptide(SAP),TEV protease was expressed as active inclusion bodies which can be purified by centrifugation.More importantly,the SAP-tagged TEV protease can be removed by centrifugation after the cleavage reaction is completed.In this study,we explored the feasibility to express P128 as ELP fusion protein and to cleave the ELP tag with SAP-tagged TEV protease,aiming at increasing P128 yield and laying the foundation for development of relevant genetic engineering products.The P128 coding sequence with a TEV protease cleavage site introduced at the 5’ end was amplified by PCR,and the PCR product was inserted into fusion expression vector pELP-Fh8.The recombinant vector pELP-Fh8-P128 was transformed into E.coli,and the conditions for ELP-Fh8-P128 fusion protein expression were optimized.The results showed that the fusion protein was correctly expressed as soluble protein.The fusion protein was purified to 90%purity by two rounds of temperature-sensitive inverse transition cycling(ITC)under optimized conditions such as phase transition temperature and salt concentration.After TEV protease cleavage and removal of ELP-Fh8 fusion tag,the recombinant P128 protein was recovered with 98%purity and the yield of 75mg/L culture.Collected 11 cases of dairy cow mastitis-related Staphylococcus aureus,Coagulase-negative staphylococcus 31 strains,Seven strains of cefoxitin were screened by susceptibility test,And then the recombinant P128 was respectively used for the bacteriostatic test,The cytotoxicity of P128 to bovine kidney cells was detected by MTT assay.The results showed that:except for 1 strain resistant to cefoxitin and Staphylococcus aureus invalid,The antibacterial activity of recombinant P128 against 9 strains of Staphylococcus aureus ranged from 0.23 and 0.94 μg/ml,The bactericidal activity was between 0.94 and 15μg/ml,and the antibacterial activity of the six coagulase-negative staphylococci was between 0.12 and 1.88μg/ml,The bactericidal activity was between 0.94 and 30μg/ml,and the activity of Streptococcus staphylococcus aureus was lower than that of susceptible strain.There is no significant cytotoxicity in bovine kidney cells.