Optimization Of HEK293 Cell Transient Expression System For Recombinant Protein Production

BackgroundThe vast majority of therapeutic recombinant proteins are produced in mammalian cell lines,among of which,Chinese Hamster Ovary(CHO)cells are an important platform for recombinant protein production.However,the proteins from CHO cells have posttranslational modifications(PTMs)that are different from human cells.Non-human PTMs can cause problems such as immunogenic responses in human cells,and the use of humanized cell lines such as human embryonic kidney cells 293(HEK293)can effectively avoid this risk.HEK293 cells have been used for transient expression of recombinant proteins,but the expression level is usually low,which is difficult to meet the application requirements.Moreover,foreign countries have a monopoly position in this field,so it is of great significance to establish and optimize the transient expression system of HEK293 with independent property rights.ObjectiveTo establish a high-efficiency transient expression system of HEK293 for recombinant protein production.Methods1.Optimization of transfection conditions and culture process: Co-transfection expression vectors containing SV40 large T antigen,p21CIP+P27cip,P18+a FGF,PDI+ Bcl-XL,XBP-1S+ERO1-La,and P18+IGF-1 were constructed,and the best auxiliary vector was screened by EGFP expression vector.Transmembrane peptides DLR8,CHR4,CPPMK2 were combined with transfection reagent in different ways,and an EGFP expression vector was used to screen out the best transmembrane peptides and the method of use.EGFP and SEAP expression vectors were used to optimize the incubation Buffer used for transfection,liquid exchange time after transfection,suspension time after transfection,selection of suspension medium and optimal concentration of p DNA.2.Optimization of bicistronic vectors:Double bicistronic vectors containing different Leader peptides were constructed and transfected into HEK293F cells.Different target genes were quantitatively or qualitatively analyzed by fluorescence microscope,flow cell number,enzyme-linked immune sorbent assay(ELISA),Western blot,and chemiluminescence.The optimal Leader and vector construction methods that can significantly enhance expression are screened out.The expression vector of the bicistronic antibody was optimized and the expression of Adalimumab was carried out using optimal transfection conditions.3.Optimization of recombinant protein expression process:Based on optimal transfection conditions,the expressions of recombinant proteins such as SEAP,HSA,and IL-6 were optimized using low-temperature culture and histone deacetylase inhibitors and optimized gene delivery vectors.Results1.The best co-transfection vector p STAB-SV40 was successfully constructed and screened,which could increase EGFP expression level by more than 3 times(P<0.001).The best transmembrane peptide DLR8 was successfully synthesized and screened,which can effectively improve the efficiency of adherent transfection and protein expression.The best optimized protocol for transfection conditions was determined.2.Two useful Leader elements were successfully selected and the optimal vector construction method was determined.48 h instantaneous expression can improve the downstream gene expression of bicistronic by about 4?10 times(P<0.05),and it is effective for different proteins.qPCR showed no correlation between m RNA level and enhanced protein expression level.The protein expression level was further enhanced by using the optimized Adalimumab expression vector based on optimal transfection conditions.3.Recombinant proteins such as HSA,IL-6,and SEAP were successfully expressed by the HEK293F cell expression system.Taking IL-6 as an example,the volume yield of IL-6 was increased by 2.26 times,3.58 times,and 5.26 times respectively(P<0.005),by co-transfer of auxiliary vector,DLR8 enhanced transfection,and sodium butyrate addition based on optimal transfection conditions.ConclusionAn efficient transient expression system for HEK293 is established by optimizing the transfer conditions,culture process,and expression vector.Then the recombinant proteins such as SEAP,IL-6,HSA,and Adalimumab have been successfully expressed.