Expression And Purification Of The Human Peptide Antibiotic HPAB-β In Pichia Pastoris And Its Biological Activity Analysis

Research background:Peptide antibiotics are small peptides encoded by organism genomic DNA. They are usually composed of 12～60 residues and are less than 10 kD. These peptides have been recognized to play important roles in the innate host defense in most living organisms. They have shown significance in controlling microorganisms before the hosts esteblish specific immunity after infected. Compared with conventional antibiotics, they have broad antimicrobial spectrums and high-performance antimicrobial activity. With the growing problem of resistance to conventional antibiotics, it has become important to study and develop peptide antibiotics.In our previous study, hPAB-βgene which encodes a beta peptide antibiotic was isolated from human keratinocytes in our laboratory, and it's antimicrobial activity was confirmed by chemosynthesis hPAB-β. In prokaryotic expression system, the fusion protein was expressed in Escherichia coli JM109, and the actual expression level of the interest peptide was less than 7%. More over, tandem multimers were designed in order to improve the procduction of the active hPAB-β, and the expression level of the interest peptide coule reach 30% after reoptimization of the production artwork.But there are still some problems in using prokaryotic expression system to produce hPAB-β. For example, through chemical cleavage, purification and renaturation, active hPAB-βcan be obtained, but the recovery rate was very low, approximately at 20%. This is not benefit for us to obtain hPAB-βby prokaryotic expression system in large-scale. So, it is necessary to find a better expression system that can immprove the expression level and obtain purier target peptide. For these considerations, the Pichia pastoris expression system was chosen to get a high expression level of hPAB-β. Research contents and results:Firstly, constructed the Pichia pastoris expression vector. Two kinds recombinant plasmids were constructed.①With 6×His tag: A: Construct recombinant pPIC9K containing natural hPAB-βsequence. B: Accoding to the code in Pichia pastoris, construct recombinant pPIC9K containing optimised hPAB-βsequence.②Without 6×His tag: Construct recombinant pPIC9K containing optimised hPAB-βsequenc.We obtained natural and optimized sequence of hPAB-βwith tag by PCR and primer extension. After digested with SnaB I and Not I, the target sequences were ligated with pPIC9K which was also digested with the same enzymes. Then, transformed the recombinant plasmids into DH5α, isolated the recombinant plasmids, and identified them by PCR, enzyme digestion and nucleotide sequencing. We obtained optimised sequence of hPAB-βwithout tag by PCR, taking optimized sequence of hPAB-βwith tag as template. After digested with SnaB I and Not I, the target sequences were ligated with pPIC9K which was also digested with the same enzymes. Then, transformed the recombinant plasmid into DH5α, isolated the recombinant plasmid, and identified it still by PCR, enzyme digestion and nucleotide sequencing.We could amplify 167bp and 144bp fragments by PCR, and the same fragments could also be released from the recombinant plasmids as expected. Nucleotide sequencing showed that the natural sequence of hPAB-βhad a samesense mutation, and the open reading frames was confirmed to be right. While the optimised sequences of hPAB-βwith/without tag and their open reading frames were both confirmed to be all right.Secondly, constructed recombinant Pichia pastoris of hPAB-β. Digested the two kinds of recombinant plasmids with Sal I, then electrotransformated them into GS115. Screened multicopy transformants by G418, and identified their phaenotypes. Then identified the transformants in levels of DNA, mRNA and protein. The results showed that 167bp and 144bp fragments coule be amplified by PCR. After inducing the transformants, 167bp and 144bp fragments could be amplified by RT-PCR.Thirdly, purification and biological activity identification of target peptides expressed in level of flask. For hPAB-βwith tag, Ni-NAT affinity chromatography was first conducted. Then P2 gel filtration chromatography was subsequently used. After that, we could obtain peptide with less impurity. For hPAB-βwithout tag, reverse phase chromatography was conducted. After purifications, aproximately 5.6kD and 4.5kD proteins could be seen by Tricine-SDS-PAGE analysis. The two kinds of recombinant hPAB-βboth showed fine bacteriostasis activity towards S.aureus and P.aeruginosa clinical isolates in the assay.Fourthly, the conditions of high-density fermentation for hPAB-β. Compared with expression in flask, the growing conditions in high-density fermentation were quite different in culture medium, cell density, dissolved oxygen, etc. So, after expression in flask, the conditions in high-density fermentation for hPAB-βwithout tag were further approached: we conducted fed-batch high-density fermentation. Inoculated engineering bacteria in BMGY medium as the first grade bateria, and then inoculated it in 200ml BMGY as the second grade bacteria. Subsequently, inoculated the latter bacteria in 2L basal salts medium into the fermentation, adding trace salts medium into it. Controlled pO2 above 70%, pH among 5.0±0.05, tempretuer at 30℃, initial speed at 600r/min. When glycerine was exhausted, and pO2 went up, add growth medium to the fermenter at 10 ml/L per hour, until the wet weight reached 200～250g/L. After 3 hours not adding growth medium into the fermenter, add induction medium into it at 3 ml/L per hour for 3 hours. Then increased the speed to 9 ml/L per hour. According to these conditions, a 4.5 kD protein was expressed in the supernatant by Tricine-SDS-PAGE analysis. The total protein concentration in 3 fermentations were 664.0μg/ml, 781.8μg/ml and 721.3μg/ml. The expression level were 11.1%, 9.8%and 10.3%. Thus, the expression quantity were 73.7mg/L, 76.6 mg/L and 74.3mg/L.Finally, the purification of recombinant hPAB-βwithout tag after high-density fermentation and biological activity identification. The fermentation supernatant was first purified by reverse phase chromatography. After that, P10 gel filtration chromatography was conducted. Tricine-SDS-PAGE analysis showed that target peptide was in peak 2. Then, RP-HPLC was finally used, and the purity of taget peptide coule reach to 95%. The purified recombinant hPAB-βshowed bacteriostasis activity towards S.aureus clinical isolate(MRSA). And it's antimicrobial activity was stronger than clindamycin and oxacillin, a little weaker than norvancomycin.Conclusion:We obtained natural and optimized sequence of hPAB-βwith 6×His tag and optimized sequence of hPAB-βwithout 6×His tag. Constructed two kinds of recombinant plasmids, screened multicopy transformants that coule express target peptides. The conditions of high-desity fermentation for hPAB-βwas groped. Then, recombinant hPAB-βwith antibacterial activity could be obtained after series of purifications.