Expression Of Porcine Follicle Stimulating Hormone In Pichia Pastoris

Porcine follicle stimulating hormone(pFSH),comprising a and ? subunits,is a glycoprotein gonadotropin synthesized and secreted by the anterior pituitary.pFSH is commonly used to induce superovulation in domestic animals in assisted reproduction technologies.However,the practical application of pFSH is inhibited by the limited efficiency,short half-life or unstable activity of its production.Due to the advantages of high yield and simple purification,Pichia pastoris expression system has been widely used for recombinant protein production of industrial interest.However,the heterologous expression efficiency of pFSH remains disappointingly low.To improve pFSH production in P.pastoris,a series of molecular strategies,together with fermentation optimization,were tested in the present study.In terms of molecular strategies,we investigated the effects of phenotypic selection of aoxl mutants,codon optimization,and fusion expression on the yield of pFSH.By comparing clones of the Muts phenotype strain,it was observed that the yield of soluble pFSH increased by approximately 96%in supernatant of Mut+ phenotype strain clones,indicating that the strains of Mut+ phenotype was more suitable for expressing pFSH.? subunit of pFSH which confers biological specificity was used to investigate the effect of codon optimization and fusion tag on the yield of pFSH recombinant protein.The results showed that the protein levels of soluble pFSH? were increased by approximately 143%and 22%after two kinds of codon optimization strategies,respectively.Moreover,the yield of pFSH? can be improved when the CAI was greater than 0.8 and G+C content more than 40%.To improve the yield of fusion protein,we investigated the effect of fusion tag,direction or linker on the yield of pFSH? protein.The results indicated that the production of fusion protein HSA-pFSH? was significantly improved compared with the production of soluble pFSH? and SUMO-pFSH?.However,the yield of fusion protein cannot be further improved by changing the orientation or linker.Moreover,we found that a large number recombinant protein was residual within the cell which cannot secrete outside,and recombinant protein was degraded at the C-terminal.In order to reduce the degradation or improve secretion efficiency of fusion protein,the YPS1 gene,which encoded protein can cleave basic amino acids in the C-terminal of recombinant proteins,was knocked out,or the signal peptide was substituted.The result indicated that knocking out YPS1 gene can reduce the production of degraded fragments in supernatant,or signal peptide HSA or MSP can effectively secrete the HSA-pFSH? protein.However,the yield of HSA-pFSH? protein in supernatant didn't increase by deletion of YPS1 gene or replacement of the signal peptide.In order to verify whether the protein of HSA-pFSH? was bioactivity,we co-expressed the gene of HSA-KRE-pFSHa on the basis of H3 strain.The optimum pH,methanol concentration of HSA-pFSH or the production in high-density fermentation were investigated.The results showed that the optimum pH and methanol concentration for expressing soluble HSA-pFSH in strain H3-3 were determined as 5.0-6.0 and 1.5-2%in shake-flask,respectively,and the yield of soluble HSA-pFSH could reach 40.8 mg/1 after purification.The yield of HSA-pFSH can reach 6 g/1 in high-density fermentation,but it accompany with degradation of a large number of proteins.Furthermore,we successfully constructed the HEK293-FSHR cell line stably expressing FSHR which can be used to detect the bioacitivity of FSH.In vitro bioactivity assays showed that recombinant HSA-pFSH could efficiently stimulate cAMP synthesis in HEK293 cells expressing porcine FSHR.And cumulus cell-oocyte complexes was expanded after dealing with HSA-pFSH,indicating that HSA-pFSH has bioactivity.In addition,we also investigated the effect of different antioxidant and co-expression molecular chaperone on the yield of HSA-pFSH?.Our results showed that the yield of HSA-pFSH? in supernatant can be improved obviously by adding antioxidant A,and the mechanism was relative to improve the secretion efficiency of HSA-pFSH?.Unfortunately,other antioxidants didn't have the similar effects,suggesting that antioxidant A increases secretion efficiency may not depend on its antioxidant activity.In addition,combined with co-expression chaperone PDI,ERO1 and BIP,antioxidant A supplementation can significantly increase the yield of HSA-pFSH? by 4-15 fold in supernatant.In conclusion,our results demonstrated that the yield of HSA-pFSH can considerably increased by 15-fold in supernatant of P.pastoris by applying molecular strategies and optimization of fermentation condition,and thus provided a valuable reference for the large-scale recombinant expression of pFSH.