Construction,Expression And Biological Activity Analysis Of Recombinant Tachyplesins ? In Genetic Engineering Strain

Tachyplesin I,originally isolated from the hemocytes of horseshoe crabs,is a 17 amino acid,cationic,antimicrobial peptide with a typical cyclic antiparallel ?-sheet structure.It exhibited potent and broad-spectrum activities against bacteria,fungi,viruses,and tumor cells.TP-I is widely regarded as a potential alternative to antibiotics and has broad application prospects in medicine,animal husbandry,food additives,and the cultivation of new crop varieties.However,limited access has resulted in no large-scale TP-I commercial products.In order to solve these problems,the tandem TP-I gene was used as the target gene,the eukaryotic expression system of Pichia pastoris with high expression of TP-I was constructed by Gene engineering,and the expression products were analyzed in various aspects.Firstly,according to the reported amino acid sequence of TP-I peptide,the relationship between TP-I structure and its bactericidal activity,the tolerance of TP-I by Pichia pastoris,etc.were analyzed,and the 17 amino acids in this paper were selected as target TP-I peptides.In order to achieve the efficient expression of selected TP-I antimicrobial peptides in engineering bacteria,a specific cleavage site(-Ala? or-Gly?)for Pancreatic elastase,and a specific cleavage site(Sequence C terminal amino acids)for carboxypeptidase A was designed.It was connected to the selected 4 peptides to form a 4-copy tandem 74 peptides.According to the principle of recombinant expressing preference codons of Pichia pastoris,the recombinant TP-I peptide gene sequence was synthesized and cloned into the highly efficient expression vector pGAPZ?B.Double enzyme digestion,PCR and sequencing analysis showed that the recombinant expression vector pGAPZ? B-4TP-I was successfully constructed.Then,the constructed expression vector pGAPZ?B-4TP-I was transformed into the host GS115 by electroporation,and the Tri-SDS-PAGE electrophoresis was used to detect the products in the fermentation solution after fermentation by YPD medium.The result of it showed that the constructed 4TP-I sequence could be expressed in the host.The fermentation broth was subjected to adsorption and elution by a Ni-IDAaffinity chromatography column,and the Tri-SDS-PAGE electrophoresis analysis was carried out through the operation of centrifugation concentrate in ultrafiltration centrifuge tube,enzymatic hydrolysis of Pancreatic elastase and carboxypeptidase.The Tri-SDS-PAGE analysis showed that there was a protein band identical to the chemically synthesized TP-I band in the vicinity of 3.3 kDa,demonstrating that the cleavage site had designed was correct.Finally,the TP-I monomer obtained by enzymatic hydrolysis was determined by BCA method and RP-HPLC method,and The content of TP-I product in the 1L shake flask fermentation broth was 27.24-29.53 mg.The TP-I monomer product was tested for bacteriostasis by Escherichia coli,Bacillus subtilis,Pseudomonas aeruginosa,and Staphylococcus aureus.The results showed that the TP-I monomer had different levels of bactericidal activity;In addition,the TP-I monomer product was entrusted to company for high-resolution time-of-flight mass spectrometry analysis,which showed that the TP-I monomer had the same molecular weight as the designed17-amino acid sequence,which was 2262.85.Therefore,the feasibility of high expression of TP-I in Pichia pastoris GS115 expression system was demonstrated.