Design Of Bovine Lactoferricin Derived Peptide And Its Expression In Pichia Pastoris

Bovine Lactoferricin(LFcinB)is a 25-residues cationic peptide,which is released from the N-terminus of bovine lactoferrin(BLf)by pepsin hydrolysis under acidic environment and its amino acid sequence is: Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-ArgMet-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile-Thr-Cys-Val-Arg-Arg-Ala-Phe.It has been proved that LFcinB have various bioactivities such as broad antibacterial activity,antitumor activity,antivirus activity,anti-parasitic and anti-oxidation activity.In addition,its thermostability and non-antigenicity are beneficial for applications in the food industry,especially the production of infant formula.Among the different LFcin from different sources,LFcinB has the highest antibacterial activity.Therefore,the research of LFcinB and its derived peptides has become one of the research hotspots in recent years.The Pichia pastoris expression system is a kind of highly efficient heterologous protein expression system.Till now,hundreds of heterologous proteins have been successfully expressed in P.pastoris.Compared to other expression systems,P.pastoris can perform post-translational glycosylation modification and processing of heterologous proteins.The secretory expression vector is linearized and integrated into Pichia pastoris genome,and the target protein is secreted into the fermentation broth by methanol-induced expression.In this paper,LFcinB was used as a template to design derivative peptides with high antibacterial activity by bioinformatics tools.The derivative peptide gene was transformed into P.pastoris genome to construct a genetic engineering strain secreting expression-derived peptide.1.In this paper,five derivative peptides were obtained by replacing the amino acids at different sites of LFcinB.Bioinformatics tools were used to predict and compare the physical and chemical parameters,hydrophobic residue ratio,charge number,amphiphilic distribution,secondary structure and spatial structure of the derived peptides.The antibacterial activity of the derivatized peptide was evaluated by the antibacterial peptide database prediction tool,and an optimized derivative peptide sequence L3 was obtained,which had a hydrophobicity of 52% and a charge number of +7.The amphipathic distribution and spatial structure simulation is similar to LFcinB.The antibacterial peptide predictive index is higher than LFcinB,and theoretically has higher antibacterial activity.2.The optimized designed derivative peptide gene was cloned into the amplification plasmid pUC-SP,and pUC-SP-L3 was transformed into competent E.coli DH5?.The target gene of L3 was obtained by double digestion.The target gene was ligated into pPIC9 K by T4 DNA ligase.Further PCR,double digestion,and DNA sequencing prove that the derived peptide gene sequence was correctly inserted into the pPIC9 K multiple cloning site.3.The recombinant expression vector pPIC9K-L3 linearized by Sac I was transformed into the Pichia pastoris GS115 competent by electroporation.The cells were applied to MD(minimum dextrose medium)plates and statically cultured at 30°C until the transformant colonies grew.41 positive phenotypes of His+Mut+ and 4 phenotypes of His+Muts were screened by selective medium MM(minimum methanol medium)and MD.PCR amplification identification indicated that the target gene has been correctly integrated into the GS115 genome.4.The 45 positive transformants obtained were screened using BMGY/BMMY medium,and the methanol concentration gradients were set at 0.5%,1.0%,1.5%,2.0%,2.5%,and 3.0%.5 positive transformers with antibacterial activity were screened,and the induced concentration was 2.5%.5.The positive transformers with the highest antibacterial activity were fermented using a 500 m shake flask.Verify and optimize expression conditions,the results of bacteriostatic experiments showed that the optimal methanol induction concentration was 2.5%,and the optimal fermentation time was 96 h.Compared with the antibacterial activity of natural LFcinB,the results showed that the antibacterial activity of the derivative peptide was better than that of the original peptide,which proved the rationality of the molecular design of the derivative peptide.Derived peptides were detected by Tricine-SDS-PAGE for efficient expression.The fermentation supernatant was freeze-dried to prepare a lyophilized powder containing the derivative peptide component.The antibacterial experiment after rehydration showed that the inhibition zone diameter of the 10× concentrate was 2.5 times that of the same volume of ampicillin solution(50 mg/mL).Based on the structure-function relationship of antimicrobial peptides,this paper used bioinformatics tools to design LFcinB-derived peptides,and analyzed its antibacterial activity.The experimental results proved the rationality of the designed peptides,and the derived peptide genes were effectively expressed.The research results laid a theoretical foundation for further expansion of the production of derivative peptides.