Krait Antibacterial Peptide Cath-BF Constitutively Secretory Expressing In Pichia Pastoris

Acne is a common skin disease of pilosebaceous inflammatory, mainly caused by anaerobic propionbacteriumacnes. Antibiotic is usually used to treat acnes, but the effect is not ideal with the risk of the drug resistance of bacteria and hypersensitivity. Antibacterial peptide Cath-BF from krait venom belongs to the first of antibacterial peptide from vertebrates. Acne model experiment in mice showed that this antibacterial peptide effectively restrained the growth of propionbacteriumacnes and other bacteria from acne, and inhibited the inflammatory hyperplasia, so it has important application value in the prevention and treatment of acne. Cath-BF separated from krait venom composed of 30 amino acid residues, but the deductive peptide from its c DNA of 34 amino acids, which posses 4 amino acid residues in the amino terminal of separated Cath-BF, but there is not experiment to prove the 34 aa peptide exists. Establishing a high effective production process of Cath-BF using gene engineering becomes urgent need because both separation from the krait venom and chemical synthesis of Cath-BF is demanding and costly. Pichia pastoris is extensively used as host cells for the expression of valuable proteins, which has good industrial performance and can effectively secrete small size peptide with the help of signal peptide from MF-α from saccharomyces cerevisiae. A constitutively secretory expression system based on the promoter of GAP and P. pastoris was developed, which makes it possible to establish a high effective production process for Cath-BF. In this study, constitutively secreting system of P. pastoris for 30 aa and 34 aa Cath-BFs were constructed, and the bacteriostatic activities of both recombinant antimicrobial peptides were tested on propionbacteriumacnes, a high effective process for fermentation and separation also established. We hope this work will played a solid foundation for the application of Cath-BF. Methods: 1. Synthesis of sequences coding Cath-BF30 and Cath-BF34Sequences coding Cath-BF30 and Cath-BF34 were designed using Pichia preferred codons, and amplified by overlap extension PCR. 2. Construction of expression vector for Cath-BF30 and Cath-BF34Coding sequences were inserted into p GAPZa, in which target peptides were fused to the 3’-terminal of MF-a to produce Cath-BF with natural amino terminal. 3.Screening and construction of recombinant Pichia secreting Cath-BF30 and Cath-BF34Expression vector plasmid DNA was linearized to electrotransformed into Pichia competent cells, high copy transformants were screened on high concentration of zeomycin YPD plates, and chose for screening high effective secreting transformants of Cath-BF. 4.Assay of the antibacterium activity of Cath-BF30 and Cath-BF34 expression productsTransformants were cultured in YPD at 30 ℃ for 72 h, and the concentrated supernatants were used as the antibacterium peptide samples. The bacteriostatic activities of samples were tested in 96-well plates containing P. acnes in anaerobic bags, OD570 of cultures were detected. 5. Effects of medium p H and culture time on the expression products of Cath-BF30 and Cath-BF34Mediums were buffered to p H4.0, 5.0, 6.0 and 7.0, respectively. 72 h cultures in these medium were used as peptide samples for the bacteriostatic activity. 6.Large-scale fermentation of Pichia pastoris Cath-BF34The fermentation of engineered bacteria was carried in 5L fermenter at p H4.0 and 25℃, fermentation supernatant was collected at 72 h. 7. Seperation of Cath-BF34 expression products SP sepharose FF, a strong cation column, was used to separate the recombinant Cath-BF34 from the fermentation supernatant. Results 1. Sequences coding Cath-BF30 and Cath-BF34 with pichia preference codon were cloned as designed. Target peptides were seamlessly fused to the C-terminus of the signal peptide MF alpha, guaranteeing the secretory product terminated at natural N-terminal sequence. 2. Recombinant Pichia strains high effectively secreting Cath-BF30 and Cath-BF34 were screened, and both expression products exhibited high activities anti-P.acne. 3. Engineering pichia cultured in the medium of p H4.0 secreted Cath-BF on the highest level, and the activity of anti-P.acne was almost undetectable when the p H of medium was above 5. These results indicated that the Cath-BF must be expressed under the condition of low p H to effectively resist the degradation by the host cell protease. 4. Batch fermentation showed that the engineering Pichia cultured in rich medium containing glucose grew to a density of 160 g/L(wet weight) and produced more than 100 mg/L Cath-BF in the supertanant. 5. The expression products of Cath-BF in fermentation supertananton were separated and purified to more than 95% purity just by a step of iron exchange on SP Sepharose FF column.ConclusionsEfficient secretion expression system for krait antibacterial peptide Cath BF30 and Cath-BF34 were successfully constructed based on constitutive promoter GAP and Pichia, and the fermentation and separation processes were also established. The expression products of both peptides showed high and same activities of anti- acne bacillus.