Enhanced Transient Recombinant Protein Production In CHO Cells Through The Co-transfection Of The Helper Genes

BackgroundChinese hamster ovary(CHO)cells are the most commonly used mammalian cell in the recombinant pharmaceutical protein expression system,and 70% of the approved recombinant proteins were derived from CHO cells.Due to low cost,short period and simple operation,transient gene expression(TGE)is not only an important tool in the early stage of biological drug development,but also widely used in scientific research.However,TGE still has some problems,such as easy loss of plasmid and low expression level.Studies have shown that the productivity of recombinant protein could be improved by the co-expression of helper genes.ObjectiveTo explore the effect of p21 CIP,p18,PDI,XBP-1s,p21CIP+p27KIP,p18+a FGF,PDI+Bcl-x L and XBP-1s+ERO1-L? co-expression on the recombinant protein expression in CHO cells.Method1.Construction of the co-transfection vectors and optimization of the co-transfection ratio.The co-transfection vectors including p21 CIP,p18,PDI,XBP-1s,p21CIP+p27KIP,p18+a FGF,PDI+Bcl-x L and XBP-1s+ERO1-L? were constructed by seamless cloning and were identified by digestion and sequencing.The gene of enhanced green fluorescence protein(eGFP)was transfected into the CHO cells as the target gene by liposome transfection,and the co-transfection ratio was 2:1,5:1,10:1 and 15:1,respectively.Cells were harvested at Day 7 and the expression level of eGFP was analyzed by flow cytometry.2.The effect and mechanism of co-transfection genes on the expression of eGFP.The eGFP and co-transfection genes were transfected into CHO cells by liposome transfection at the ratio of 10:1.Then,the eGFP expression was detected by the inverted fluorescence microscope after 48 h transfection.And the expression level of eGFP was analyzed by flow cytometry,Western Blot and q PCR at day 7,respectively.The viable cell density and cell viability were analyzed at day 1,3,5,6 and 7.Cell cycle and apoptosis were analyzed by flow cytometry at day 4 and day 7,respcetively.3.The effect of co-transfection genes on the expression of adalimumab and vitronectin.The adalimumab or vitronectin vectors and co-transfection vectors were transfected into CHO cells by liposome transfection at the ratio of 10:1.The supernatant was collected Day 7 and the expression level of adalimumab or vitronectin in CHO cells were analyzed by ELISA.Result1.The eight co-transfection vectors containing different co-transfection genes were successfully constructed.With the co-transfection ratio of 2:1,the expression level of eGFP was improved by 1.34 fold by XBP-1s.With the ratio of 5:1,the expression level of eGFP was improved by 1.13 fold by p21 or p18+a FGF.With the ratio of 15:1,the expression level of eGFP were improved by 1.13,1.16,1.23 or 1.14 fold by PDI,XBP-1s,p21+p27 or p18+a FGF,respectively.The expression level of eGFP were all increased by 1.43 to 2.02 fold with the co-transfection genes at the ratio of 10:1.Therefore,the optimal co-transfection ratio of target plasmid and co-transfection plasmid was 10:1.2.After co-transfected with eGFP and co-transfection genes with the ratio of 10:1,the expression level of eGFP were increased by 1.72,1.81,1.67,1.54,1.51,2.02,1.43 or 1.79 fold,respectively,but were not connected with m RNA expression levels.The percentage of G1 phase cells was increased by 6.19%,4.52%,5.27% or 2.26% by p21,p18,p21+p27 or p18+a FGF,respectively.Co-expression of PDI or XBP-1s increased the ratio of oxidized to reduced forms of VTN protein by 1.14 or 1.4 times,respectively.3.The expression levels of adalimumab were increased by 1.32,1.31 or 1.57 fold after co-transfected with p21,XBP-1s or PDI+Bcl-x L,respectively.The expression levels of vitronectin were increased by 1.22,1.19 or 1.34 fold after co-transfected by PDI,XBP-1s or p21+p27,respectively.ConclusionThe expression of adalimumab was increased by overexpression of p21,XBP-1s or PDI+Bcl-x L.The expression of vitronectin was increased by overexpression of PDI,XBP-1s or p21+p27.However,the expression of recombinant proteins were not improved by other co-transfection genes.Further research,the increased gene expression by p21 or p21+p27 may be associated with cell cycle arrest and the inhibition of cell growth.And the increased protein expression by PDI or XBP-1s may be related to the promotion of oxidative folding of proteins.