Mytilus contain abundant antimicrobial peptides(AMPs)that play a key role in the innate immunity.However,heterologous production of these AMPs remains challenging due to their short sequences,multiple disulfide bonds,...Mytilus contain abundant antimicrobial peptides(AMPs)that play a key role in the innate immunity.However,heterologous production of these AMPs remains challenging due to their short sequences,multiple disulfide bonds,and high content of cationic amino acids,which hinder functional expression in prokaryotic systems such as Escherichia coli.To establish a eukaryotic recombinant expression system for the AMPs of mussel and obtain recombinant mussel AMPs for subsequent studies,we reported the successful recombinant expression of myticofensin B1,a novel defensin-like AMP identified previously in Mytiluscoruscus,using the eukaryotic host Pichia pastoris.The codon-optimized gene encoding the mature myticofensin-B1(composed of 65 amino acid residues,including 6 conserved cysteine residues)was cloned into a pPICZαA vector and expressed in P.pastoris GS115.Structural fidelity of the recombinant peptide was confirmed by liquid chromatography-tandem mass spectrometry(LC-MS/MS),showing a molecular weight of 8849.9 Da,which was consistent with the theoretical prediction.Functional assays demonstrated a broad-spectrum antimicrobial activity of the recombinant myticofensin-B1,with stronger inhibition against Gram-negative bacteria.Scanning electron microscopy revealed different effects of the recombinant myticofensin-B1 against different bacteria.In addition,the recombinant myticofensin-B1 exhibited a very low hemolytic activity against sheep red blood cells and weak cytotoxicity against human A549 lung cancer cells.This study establishes P.pastoris as a powerful platform to produce functional mussel AMP and highlights the potential of the recombinant myticofensin-B1 as a therapeutic agent for aquaculture pathogens and infections.展开更多
[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was ...[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sarr~ was integrated into Pichia pastoris GSl15 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-pedormance liquid chroma- tography （HPLC） by determining the production of S-adenosy-L-methionine （SAM） with the enzyme secreted. [ ResultJ The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosytation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, espe- cially the latter, after which the specific activity of SAMS was improved to 61.48 U/rng. [Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GSl15 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.展开更多
[Objective] This study aimed to investigate the secretory expression of P97R1 gene of Mycoplasma hyopneumoniae（Mhp） in Pichia pastoris expression system and the primary application; of the expression product. [Metho...[Objective] This study aimed to investigate the secretory expression of P97R1 gene of Mycoplasma hyopneumoniae（Mhp） in Pichia pastoris expression system and the primary application; of the expression product. [Method] A pair of specific primers was designed to conduct PCR according to the Mhp P97R1 gene sequence in Genbank, and the amplified P97R1 gene was cloned into the pPICZa-A yeast expression vector to construct the secretory recombinant expression vector pPICZa-A-P97R1. The plasmid pPICZa-A-p97R1 linearized by Sac I was transformed into P. pastoris GSl15 by electroporation. Positive transformant identified by PCR was incubated to express P97R1 protein after methanol induction. And the expression product was identified using SDS-PAGE and Western-blotting anal.wsis. [Result] P97R1 protein was successfully expressed in the P. pastoris system, with a secre- tory amount of 499μg/ml, and revealed good reactogenicity. Meanwhile, an indirect ELISA method was established with P97R1 protein after the optimization of each reaction factor, which showed good specificity and repeatability according to repeated tests. [Conclusion] This study provides bases for developing the ELISA Kit for anti- body detection and genetically engineered vaccine to Mhp.展开更多
基金supported by the National Natural Science Foundation of China(32271580,42020104009)the Fundamental Research Funds for Zhejiang Provincial Universities and Research Institutes(JX6311101923)。
文摘Mytilus contain abundant antimicrobial peptides(AMPs)that play a key role in the innate immunity.However,heterologous production of these AMPs remains challenging due to their short sequences,multiple disulfide bonds,and high content of cationic amino acids,which hinder functional expression in prokaryotic systems such as Escherichia coli.To establish a eukaryotic recombinant expression system for the AMPs of mussel and obtain recombinant mussel AMPs for subsequent studies,we reported the successful recombinant expression of myticofensin B1,a novel defensin-like AMP identified previously in Mytiluscoruscus,using the eukaryotic host Pichia pastoris.The codon-optimized gene encoding the mature myticofensin-B1(composed of 65 amino acid residues,including 6 conserved cysteine residues)was cloned into a pPICZαA vector and expressed in P.pastoris GS115.Structural fidelity of the recombinant peptide was confirmed by liquid chromatography-tandem mass spectrometry(LC-MS/MS),showing a molecular weight of 8849.9 Da,which was consistent with the theoretical prediction.Functional assays demonstrated a broad-spectrum antimicrobial activity of the recombinant myticofensin-B1,with stronger inhibition against Gram-negative bacteria.Scanning electron microscopy revealed different effects of the recombinant myticofensin-B1 against different bacteria.In addition,the recombinant myticofensin-B1 exhibited a very low hemolytic activity against sheep red blood cells and weak cytotoxicity against human A549 lung cancer cells.This study establishes P.pastoris as a powerful platform to produce functional mussel AMP and highlights the potential of the recombinant myticofensin-B1 as a therapeutic agent for aquaculture pathogens and infections.
文摘[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sarr~ was integrated into Pichia pastoris GSl15 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-pedormance liquid chroma- tography （HPLC） by determining the production of S-adenosy-L-methionine （SAM） with the enzyme secreted. [ ResultJ The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosytation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, espe- cially the latter, after which the specific activity of SAMS was improved to 61.48 U/rng. [Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GSl15 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.
基金Supported by the National Natural Science Foundation of China(31100136)the Special Fund for Independent Innovation of Agricultural Science and Technology in Jiangsu Province[CX(12)5051]~~
文摘[Objective] This study aimed to investigate the secretory expression of P97R1 gene of Mycoplasma hyopneumoniae（Mhp） in Pichia pastoris expression system and the primary application; of the expression product. [Method] A pair of specific primers was designed to conduct PCR according to the Mhp P97R1 gene sequence in Genbank, and the amplified P97R1 gene was cloned into the pPICZa-A yeast expression vector to construct the secretory recombinant expression vector pPICZa-A-P97R1. The plasmid pPICZa-A-p97R1 linearized by Sac I was transformed into P. pastoris GSl15 by electroporation. Positive transformant identified by PCR was incubated to express P97R1 protein after methanol induction. And the expression product was identified using SDS-PAGE and Western-blotting anal.wsis. [Result] P97R1 protein was successfully expressed in the P. pastoris system, with a secre- tory amount of 499μg/ml, and revealed good reactogenicity. Meanwhile, an indirect ELISA method was established with P97R1 protein after the optimization of each reaction factor, which showed good specificity and repeatability according to repeated tests. [Conclusion] This study provides bases for developing the ELISA Kit for anti- body detection and genetically engineered vaccine to Mhp.
