Design, Expression And Purification Of Tandem Multimers Of Peptide Antibiotic HPAB-β

Peptide antibiotics are small peptides encoded by organism genomic DNA. They have been recognized to play important roles in the innate host defense of most living organisms and their broad antimicrobial spectrums and strong antimicrobial activities position them as imaginative weapons for fighting bacterial and fungal infections. The growing problems of resistance to conventional antibiotics and the need for new antibiotics have stimulated the interests in the development of peptide antibiotics as human therapeutics.In our previous study, a sequence encoding a protein PaP3.30(16.7kD) selected out from Pseudomonas aeruginosa phage 3 (PaP3) was used as carrier molecule to coexpress with hPAB-p gene 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-p (4.3kD) was only one-sixth of that of the fusion protein(23.6kD), and the actual expression level of the interest peptide was less than 7%.In order to improve the production of the active hPAB-p peptides effectively, tandem multimers of hPAB-p gene were constructed and the following experiments were conducted:Firstly, design, construction and expression of the tandem multimers of hPAB-p gene. A pair of primer was designed for amplification of hPAB-p gene by PCR. In this primer pair, two restriction enzyme sites, BamHI(GGATCC) and ,Ava(ATGCAT), and one hydroxylamine cleavage site(AsnGly) were added to the 5' terminal of the forward primer; and the other two restriction enzyme sites, Hind(AAGCTT) and Pst\ (CTGCAG), and a hydroxylamine cleavage site were introduced to the reverse primer.The PCR products were purified and cloned into pUCni-T vectors to screen pUCm-T-hPAB-p. The Bam/Hind digested target fragments from pUCm-T-hPAB-p were subcloned into pQE-31 vectors to get the recombinants pQE-hPAB-p (1), which bearing 1 copy of peptide antibiotic hPAB-p gene. An identical cohesive end (TGCA) would occur when digested pQE-hPAB-p(l) with Ava(ATGCAT) and PstI(CTGCAG), after digestion separately, the new ligation (ATGCAG) would be recognized by neither of the enzymes. Based on the properties of Ava and PstI, the larger fragments from pQE-hPAB-p (1) digested with XhoI/Ava and the smaller fragments digested with XhoI/PstI (both fragments containing 1 copy of hPAB-p gene) were ligated to produce the recombinant plasmids pQE-hPAB-p (2), which harboring 2 copies of hPAB-p gene. Practically, pQE-hPAB-p (3~8) were also successfully constructed by ligating the proper larger and smaller fragments obtained with XhoI/Ava and XhoI/PstI digestions. All positive recombinants were identified by Bam/Hind digestions and pQE-hPAB-p (1 ~8) could respectively release about 154, 292, 430, 568, 706, 844, 982 and 1120bp fragments as expected. The target sequences and the open reading frames of the recombinants were also confirmed to be right by direct DNA sequencing. The engineered bacteria harboring pQE-hPAB-(l~8) induced by 0.5mM IPTG could express the expected tandem fusion proteins. The antigenicities of the expression bands were clarified by Western blotting using antibodies to synthetic hPAB-p peptides.Secondly, screening of the genetic engineered bacteria. In order to determine which of the engineered bacteria is the optimal one for the preparation of peptide antibiotic hPAB-p, the candidates containing pQE-hPAB-p(l~8) were induced to express tandem multimers separately and analyzed by Tricine-SDS-PAGE. Their expression levels were 2.6%, 19.7%, 27.8%, 28.0%, 24.5%, 10.2%, 7.9% and 5.7% respectively. The cellular yields (cell weights) of the bacteria whose expression levels 19.7% were also counted, there were 2.192, 3.153, 1.747 and 2.424 g/L for 2-5 multimers produced bacteria respectively. All target multimers were expressed as inclusion bodies in E. coli, but only the inclusion bodies from 2 or 3 copies engineered bacteria could be completely dissolved in 8M urea and the proteins of interest could also be ca...