Expression Of Fish Antimicrobial Peptides In Pichia Pastoris

Fish living in water environment is easy to encounter a variety of pathogenic microorganism infection.And their body has a stronger immune system than other organisms to resist this complex living environment.This super immune system benefits from having many natural antibacterial peptides(AMPs)in their body,such as lysozyme and defensin.Lysozyme(EC3.2.1.17)is hydrolytic activity against bacterial peptidoglycan and plays an important role in the innate immune system of all animals including fish.Defensin is some small molecule peptides and an important part of animal immune defense system.This study focuses on fish lysozyme and ?-defensin.At present,it needs to use a lot of antibiotics and preservatives in aquaculture industry.If the natural antimicrobial peptides can partly replace the use of antibiotics and preservatives,it is very significant to improve the quality and increase the economic efficiency for aquatic products.But the content of natural antimicrobial protein is low in organism,and the antibacterial effect in vitro is limited.Therefore,based on the natural antimicrobial peptides from fish,the hybrid antibacterial peptides were designed to increased potency.In the mean time,DNA recombinant expression was used to produce antimicrobial peptides.That is in line with the current needs of aquatic breeding and processing industries.This paper mainly studied the expression of common carp(Cyprinus carpio)g-type lysozyme and the hybrid antimicrobial peptide from channel catfish(Ictalurus punctatus)c-type lysozyme fused with zebrafish(Danio rerio)?-defensin in Pichia pastoris.The g-type lysozyme plays an important role in the fish's immune response against microbial invasion.In the present study,a new g-type lysozyme isoform gene(CLYG)was separated from common carp gill.The full-length cDNA of CLYG is 558 bp,which encoded 185 amino acid residues.The amino acid sequence deduced from the cDNA of CLYG did not contain a signal peptide and possesses three catalytic residues(Glu73,Pro86 and Asp97),which was similar to the g-type lysozyme from other teleost species.According to the align results of the amino acid sequence of g-type lysozyme between common carp and other organisms,it was found that the g-type lysozyme in common carp gill has the highest homology with the g-type lysozyme in common carp head kidney.Xho I and Xba I restriction sites were added to 5' and 3' ends of the CLYG by PCR,respectively.The expected fragment was ligated to pPICZ?A vector to construct a recombinant expression vector pPICZ?A-CLYG.Colony PCR,restriction enzyme digestion with XhoI and XbaI,and sequencing confirmed that pPICZ?A-CLYG recombinant vector was successfully constructed.Then the pPICZ?A-CLYG was transformed into competent P.pastoris X-33 cells.Recombinant CLYG was induced with 1.0%(V/V)methanol at 29?,pH 6.0 for 72 h.SDS-PAGE analysis and Western blot analysis demonstrated that recombinant CLYG was expressed.Zebrafish ?-defensin is small cationic antimicrobial peptides containing six conserved cysteine residues that form three intramolecular disulfide bonds.Channel catfish c-type lysozyme possesses two catalytic residues(Glu50 and Asp67),and eight conserved cysteine residues that form four intramolecular disulfide bonds.?-defensin and c-type lysozyme has synergistic effect to enhance the antibacterial activities against invading microorganisms and they are suitable for fused expression.In the present study,codon optimized cDNA from channel catfish c-type lysozyme mature peptide(lyc)and zebrafish defensin beta-like 3 mature peptide(defbl3)were connected by SOE-PCR to development as a novel hybrid antimicrobial agent(LD)with increased potency in fish.Using XhoI and XbaI restriction sites,the expected fragment was ligated to pPICZ?A vector to construct a recombinant expression vector pPICZ?A-LD.Between the sequence of lyc and defbl3,4×Gly flexible linker and enterokinase cleavage site were designed.Recombinant expression vectors pPICZ?A-LDn(n=1,2 or 4)harboring 1-,2-,and 4-copy LD expression cassette were constructed and confirm by digestion with BglII and BamHI.Vectors linearized by Bgl II were transformed into competent P.pastoris X-33 cells by electroporation.The gene copy number of the transformants was quantified by real-time quantitative PCR.The result is indicated that the construction of multiple copy expression cassettes can indeed increase the copy number of the target gene integrated into the yeast genome.The target protein LD was purified by affinity column after 120 h induction with 0.75%(V/V)methanol.SDS-PAGE analysis and Western blot analysis showed that the target protein LD was expressed.X-33/p PICZ?A-LD4 obtained the highest recombinant LD yield,which is the 1.67 times higher than the expression level of X-33/pPICZ?A-LD.It confirmed the strategy that constructing vectors harboring multiply copied target gene expression cassette is effective for improving the LD expression level in recombinant P.pastoris system.By monitoring the growth of yeast cells,it was found that the hybrid antibacterial peptide LD was more suitable than defbl3 when they were expressed in P.pastoris.Antibacterial assays showed that the fermentation supernatant containing the recombinant LD had bioactivities against Gram-positive bacteria Listeria monocytogenes and Gram-negative Pseudomonas aeruginosa.In this study,we successfully expressed recombinant g-type lysozyme and recombinant hybrid antibacterial protein LD in P.pastoris expression system.This study lays a foundation for further understanding of protein properties and functions of ?-defensin and lysozyme,and also created the conditions for its development and application.