Reoptimization Of The Production Artwork And Initial Study On The Biological Activities Of Human Peptide Antibiotic β

Peptide antibiotics are small peptides encoded by organism genomic DNA. They have been recognized for their antimicrobial activities in the innate host defense of most living organisms. Most of these peptides are constitute of 12～60 residues and less than 10 kDa. Their broad antimicrobial spectrums and strong bacteriocidal activities position them as imaginative weapons for fighting drug-resistance bacterial and fungal infections in future.In past five years, a sequence encoding a protein PaP3.30 (16.7kDa) selected out from Pseudomonas aeruginosa phage 3 (PaP3) was used as a carrier molecule to coexpress with hPAB-βgene which is a beta peptide antibiotic isolated from human keratinocytes by our laboratory. The expressed fusion proteins reached 40% of the total Escherichia coli JM109 proteins. But the molecular weight of the target peptide hPAB-β(4.3kDa) was only one-sixth of that of the fusion protein (23.6kDa), and the actual expression level of the interest peptide was less than 7%. In order to improve the production of the active hPAB-βeffectively, its recombinant vectors bearing 1~8 copies of hPAB-βgene in tandem multimers respectively were successfully constructed by isocaudaners AvaIII and PstI and these plasmids could express the desired tandem multimers in E. coli JM109. The 3 multimers produced bacterium was screened out as the best candidate for the preparation of rhPAB-βbased on the total cell yields, the expression level (20%~30%) of the target fusion proteins and the solubility of the fusion proteins in urea. The optimal parameters of the recombinant bacteria fermentation in the bottle and fermenter were determined. When the expressed 6×His-tagged fusion protein captured by affinity chromatography, the 3 multimers fusion proteins could be cleaved by hydroxylamine and the hPAB-βmonomers could retain their antibacterial activities after...