Expression Of Porcine Pepsin In Pichia Pastoris And The Effect Of Trna Abundance On The Expression Of Exogenous Gene

Porcine pepsin is an acidic hydrolase,which has a wide range of applications in feed,food,leather,medicine,etc.This experiment successfully integrated pig pepsin into the Pichia pastor is GS115 genome and successfully expressed in Pichia pastoris.Expression,optimization of the fermentation conditions,obtained in the shake flask level of pepsin in BMGY-1 medium expansion culture,in the BMMY-1 medium to add methanol to a final concentration of 2.5%to induce expression,reached the maximum enzyme on the ninth day 2322 IU/mL;Pichia pastoris strain 10 L fermentation tank expansion culture,pig pepsin enzyme activity up to 11388 IU/mL,compared with shake flask fermentation enzyme activity increased by 4.9 times.Translocation ribonucleic acid(tRNA)is one of the important components in protein synthesis.In order to explore the effect of the changes of tRNAs corresponding to rare codons(rarity tRNAs)on the expression of exogenous genes,the co-expression system of rare tRNA gene and exogenous gene in Pichia pastoris was constructed.The expression of GFP in Pichia pastoris can be greatly reduced when a repressor region composed of four continuous proline rare codon CCG was added into the GFP gene.The expression amount of the repressed GFP could be increased about 4.9%when tRNACCGPro gene was cointegrated to the 3' of the repressed GFP gene through pPIC9K to the genome of Pichia pastoris GS115.Meanwhile,the expression amount of the repressed GFP increased about 12.5%by integrating the repressed GFP gene and tANACCGPro gene to the genome of Pichia pastoris GS115 through pPIC9K and pFLDa,respectively.Using the same method,NFATc3T-GFP fusion gene and tRNACCGPro gene were co-expressed in Pichia pastoris GS115 resulting in 21.3%increased of the expression amount of NFATc3T-GFP fusion protein.In conclusion,tRANACCGPro gene has been confirmed to be a kind of rare tRNAs in Pichia pastoris GS115.Through co-expression of tRNACCGPro gene and heterologous genes which containing the continuous rare codon CCG,the expression of the repressed heterologous genes could be increased significantly.Furthermore,this co-expression system would contribute to screening and determining the other rare tRNAs.At the same time,it was verified that the co-expression of added tRNAs in the minimal medium induced expression was more obvious than in the rich medium.