The low-expression level of lactoferrin(LF)in the production process poses a significant challenge.This study aimed to effi-ciently express bovine lactoferrin(BLF)using Pichia pastoris GS115 as the expression host and...The low-expression level of lactoferrin(LF)in the production process poses a significant challenge.This study aimed to effi-ciently express bovine lactoferrin(BLF)using Pichia pastoris GS115 as the expression host and PIC9K as the recombinant vector.Optimization strategies included codon usage,promoter selection,and fermentation conditions.The blf gene was optimized for P.pastoris GS115 bias,resulting in the construction of the recombinant vector pPIC9K-UBLF1-3 controlled by the AOX1 promoter.SDS-PAGE analysis revealed soluble and efficient expression of ublf3 in P.pastoris GS115,with a molecular mass of approximately 76 kDa.The transformant P.pastoris GS115/pGAP9K-UBLF3-4 resistant at 4 mg·mL^(−1)G418,exhibited a ublf3 gene copy number of 5.88 through high-copy screening.Optimal expression conditions of recombi-nant UBLF were determined as 24℃,pH 5.0 and 220 r·min^(−1)through fermentation condition optimization.Under these con-ditions,recombinant UBLF production reached 40.62 mg·L^(−1).The yield of recombinant UBLF was reached 824.93 mg·L^(−1)through high-density fermentation.Antibacterial assay demonstrated the efficacy of recombinant UBLF against Escherichia coli JM109 and Staphylococcus aureus CGMCC 1.282.This study successfully achieved the efficient heterologous expression of recombinant UBLF in P.pastoris GS115,providing valuable insight for industrial production and the potential develop-ment of natural antibacterial agents.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 Key Research and Development Program of China(2023YFA0914500)the National Science Foundation of China(32271487)+1 种基金the National First-class Discipline Program of Light Industry Technology and Engineering(LITE2018-12)the Program of Introducing Talents of Discipline to Universities(111-2-06).
文摘The low-expression level of lactoferrin(LF)in the production process poses a significant challenge.This study aimed to effi-ciently express bovine lactoferrin(BLF)using Pichia pastoris GS115 as the expression host and PIC9K as the recombinant vector.Optimization strategies included codon usage,promoter selection,and fermentation conditions.The blf gene was optimized for P.pastoris GS115 bias,resulting in the construction of the recombinant vector pPIC9K-UBLF1-3 controlled by the AOX1 promoter.SDS-PAGE analysis revealed soluble and efficient expression of ublf3 in P.pastoris GS115,with a molecular mass of approximately 76 kDa.The transformant P.pastoris GS115/pGAP9K-UBLF3-4 resistant at 4 mg·mL^(−1)G418,exhibited a ublf3 gene copy number of 5.88 through high-copy screening.Optimal expression conditions of recombi-nant UBLF were determined as 24℃,pH 5.0 and 220 r·min^(−1)through fermentation condition optimization.Under these con-ditions,recombinant UBLF production reached 40.62 mg·L^(−1).The yield of recombinant UBLF was reached 824.93 mg·L^(−1)through high-density fermentation.Antibacterial assay demonstrated the efficacy of recombinant UBLF against Escherichia coli JM109 and Staphylococcus aureus CGMCC 1.282.This study successfully achieved the efficient heterologous expression of recombinant UBLF in P.pastoris GS115,providing valuable insight for industrial production and the potential develop-ment of natural antibacterial agents.
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.
