Purification Of Peptide Antibiotic Hpab-β Expressed In Pichia Pastoris And Its Effects On Herpes Simplex Virus

Peptide antibiotics are small peptides (usually composed of 12-60 residues and<5 kDa) encoded by the genomic DNA of organisms. The widespread distribution throughout the plant and animal kingdoms implied that peptide antibiotics have played a key role in the growth of complex higher organisms. Although some anionic antimicrobial peptides are discovered, but the most found peptide antibiotics are cationic. Defensins are a family of cationic peptide antibiotics firstly found in rabbit polymorphonuclear neutrophils (PMN) and then in a number of phagocytes, paneth cells, epithelialis from other mammals including humans, which enable the host to fend off the invasion of a wide range of microbes, such as bacteria, fungi, and viruses. Based on the spatial distribution of the cysteine residues, mammalian defensins are divided into three subfamilies:theα-,β-, andθ-defensins. Human defensins contain a conserved motif of six intralinked cysteines forming three S-S bridges. To date, at least 17 human defensin have been discovered:sixα-defensins (HNP1-4, HD5 and HD6) and 11β-defensins (HBD1-6 and HBD25-29). Peptide antibiotics have shown significance in controlling microorganisms before the establishment of host specific immunity when infected. They are promising antibiotics with low susceptibility to acquired bacterial resistance because of their unique mechanism in antimicrobial action. The emergence of multiple-drug-resistant bacteria, fungi and parasites in recent two decades has fueled considerable interest in using endogenous antibiotic peptides as alternatives to commonly used antibiotics derived from the microbial world. In the process of developing antibiotic peptide as a new therapeutical agent, one of the great obstacles is the production of peptide of interest in large scale.The hPAB-βwas derived from human beta-defensin 2 (hBD-2) by missing the threeThis work was supported by grants from the National Natural Science Foundation of China (no. 30772061), Natural Science Foundation of Chongqing City (no. CSTC,2005AB5201) and The "Eleven-Five" project of PLA (no.06G075). N-terminal amino acids and adding an asparagine to its C-terminal. The first pure hBD-2 was isolated from psoriatic scale extracts using a whole Escherichia coli affinity column. The expression of hBD-2 is greatly upregulated with tumor-necrosis factor-a within 1 h of stimulation and persisted for more than 48 h, Gram-negative and Gram positive bacteria strongly induce hBD-2, implied that hBD-2 plays critical role in protection of human epithelia and mucosa from the infection. In our previous study, the heterologic expression of hPAB-βin E. coli was performed by using a 16.7 kDa protein from Pseudomonas aeruginosa phage 3 (PaP3, GenBank accession number:AY078382) as a carrier molecule. The expressed fusion protein reached 40% of total E. coli JM109 proteins, while the calculated molecular weight of the target peptide hPAB-β(4.2 kDa) was only about one-sixth of that of the fusion protein (23.6 kDa), and the actual expression amount of interested peptide was less than 7%. In order to improve the production of active hPAB-βeffectively, its tandem multimers were designed and only hPAB-βtrimer fusion protein inclusion bodies recovered from the induced E. coli extracts was able to dissolve in 8M urea completely. The chemical cleavage efficiency (cleaved hPAB-βtrimer into monomers by hydroxylamine treatment) and recovery rate were also very low. For the above considerations, the Pichia pastoris expression system was chosen. The coding sequence of hPAB-βwas firstly optimized according to the codon usage bias of yeast and the untagged hPAB-βrecombinant pPIC9K were constructed and choosed for the expression of peptide antibiotics hPAB-β. In this desertation, the high-cell density fermentation and purification of hPAB-βwere performed and the antiviral activity of hPAB-βagainst herpes simplex virus (HSV) was also evaluated. The major methods and results are as follows:The high-cell density fermentation of pPIC9K-hPAB-β/GS115 strain You5 was performed in a 3.7 L fermentor. The expression of the recombinant hPAB-βwas induced by the addition of methanol. The growth curve and the expression level of hPAB-βwere determined during fermentation. The results show that the cells grew faster among the first 16h and continued to grow by the addition of the supplemental culture. In 32h post inoculation, the weight of the wet cells reached 236.7±25.3g/L and the methanol was added to induce the expression of the target gene. The supernatant was taken in 8-12h intervals and subjected to detect the protein of interest with Tricine SDS-PAGE. The expected peptide can be detected in 12h post induction and gradually enriched with the induction time. The fermentation was stoped 60h post induction and the total proteins in the fermentative supernatants were 664.0 mg/L,781.8 mg/L and 721.3mg/L respectively (3 times of fermentation) determined by Brandford assay. The hPAB-βreached 33.4% of the supernatant total proteins analyzed by Quantity-one software (Bio-Rad) and the calculated amounts of expressed hPAB-βwere 241.2±29.5 mg/L.The aim of the purification is not only to get the pure products, but also to keep the bioactivity of the protein during the process of purification. The hPAB-βis a small cationic peptide with a pI of 9.001, so we designed to use lOkDa membrane ultrafiltration, reverse phase chromatogryphy, cation exchange and gel filtration to purify hPAB-βfrom the fermentative supernatants. The final recovery was 74.9±1.5 mg/L with a purity of 98%. The overall recovery rate was 31.5% after a 4-step purification process. The purified hPAB-βcould react with the antibodies against the chemical synthetic hPAB-βin a western blot assay and retained its bactericidal activity against Staphylococcus aureus and Pseudomonas aeruginosa. These results showed that the methylotrophic yeast-inducible system produced hPAB-βcan be purified to a high purity with lOkDa membrane ultrafiltration, reverse phase chromatogryphy, cation exvhange and gel filtration process and the purified hPAB-βcould retain its bioactivity similar to that of the synthetic hPAB-β.The viral isolation is a golden standard in the diagnosis of genital herpes and usually takes 2-4 days. The positive rate of viral isolation depends on the origins of the clinical samples. PCR is a sensitive molecular biological technique used for the diagnosis of many infectious diseases. The primers were designed based on the gene sequence of the DNA polymerase of HSV and used to detect 60 clinical samples taken from the suspect patients with genital herpes. Forty-five samples were positive (75%) detected with the type specific primers for HSV-2, and no positive for HSV-1. Among the 60 samples,36 were positive (60%) in the viral isolation using Vero cells. The positive rate of viral culture was significantly lower than that of the PCR detection (X2=24.38, P<0.01). The successful detections mainly came from the samples of blisters and pustules (with a positive rate of 80-93.3% in both PCR detection and viral isolation). The anabrosis, incrustation and maculopapule samples were offen with lower detection rate. These results implied that the blister and pustule samples are suitable for the diagnosis of patients with genital herpes.The peptide antibiotics hPAB-βwas prepared by performing high-cell density fermentation with recombinant pPIC9K-hPAB-β/GS115 Pichia pastoris. The cell toxicity of hPAB-βagainst Vero cells was determined by serial dilution assay and the. results show that the high concentration of hPAB-β(≥1mmol/L) has toxicity to Vero, the highest notoxicitic concentration is 400μmol/L, about 1720μg/ml. The 400-10μmol/L hPABβhave antiviral activity against HSV-2, the numbers of viral plaques decreased 20%-79% after 1.5h treatment with hPAB-βand then inoculated into Vero cells for 2d culture. In the control group,100TCID50 of HSV-2 solution 0.2ml could form 101.7±5.5 plaques in Vero cells after 2d culture and stained by 1% crystal violet. In the similar experiment condition, only the high concentration of acyclovir (>100μmol/L) could be detected its antiviral activity.After treated with 200μmol/L acyclovir, the viral plaques forming was 39.0% of that from virus control group. Likewise, the rate of viral plaques forming was 48.7% after treated with 200μmol/L of peptide antibiotics hPAB-β. The rates of viral plaques forming decreased significantly in both acyclovir (P=0.0103) and hPAB-β(P=0.02545) treated group comparison to the viral control, but there was no significant difference between acyclovir and hPAB-βtreatment (P=0.06927). The results implied that the peptide antibiotic hPAB-βhas favourable antiviral activity against HSV-2.In conclusion, the expression levels of peptide antibiotic hPAB-βwere 241.2±29.5 mg/L in high-cell density fermentation with recombinant Pichia pastoris. The final recovery of hPAB-βfrom the fermentative supernatants was 74.9±1.5 mg/L after a four-stepwise purification process (lOkDa membrane ultrafiltration, reverse phase chromatogryphy, cation exchange and gel filtration). The overall recovery rate was 31.5%. The purified hPAB-βretained its bactericidal activity against Staphylococcus aureus and Pseudomonas aeruginosa. Sixty clinical samples taken from the suspect patients with genital herpes were studied by PCR detection and viral isolation with Vero cells. Forty-five samples were positive (75%) in PCR detection with the type specific primers for HSV-2, and no positive for HSV-1. Among the 60 samples,36 were positive (60%) in the viral isolation using Vero cells. The positive rate of viral culture was significantly lower than that of the PCR detection. The antiviral activity of hPAB-βagainst HSV-2 was evaluated by viral plaques counting assay. The plaques forming rates were 48.7% and 39.0% after treated with 200μmol/L hPAB-βand acyclovir respectively, but there was no significant difference between hPAB-βand acyclovir treatment group. Our results show that the hPAB-βexpressed in recombinant Pichia pastoris has favourable antiviral activity, which lay down a good foundation for futher analysis of the mechanism of hPAB-βagainst the virus.