Establishment Of APRT-deficient CHO Cell Line And Its Effect On The Expression Of Recombinant Protein

BackgroundIn the biopharmaceutical industry,Chinese hamster ovary(CHO)cells are the most widely used mammalian cell expression system for recombinant drug protein production,and the variation in transgene expression levels between CHO cell clones has led to a prominent problem of instability(heterogeneity)in recombinant protein expression.Adenine phosphoribosyltransferase(APRT),a key enzyme in the purine remediation pathway of ATP synthesis in organisms,is not only involved in the anabolism of purines in organisms,but also has an important impact on gene expression caused by gene silencing.Therefore,this study focused on obtaining aprt gene knockout CHO cell lines and investigating their effects on recombinant protein expression through screening,with the aim of eventually establishing a gene amplification system based on APRT-deficient cell lines for the efficient expression of target proteins.ObjectiveThe aim of this paper is to knocked out the adenine phosphoribosyltransferase(APRT)gene of Chinese hamster ovary(CHO)cells using CRISPR/Cas9 gene editing technology and to investigate the effect of the obtained APRT-deficient CHO cell line on the expression of recombinant protein.Methods(1)Aprt gene knockout and validation.The sg RNA was designed and synthesized according to the aprt genome sequence of CHO cells,and the aprt gene was knocked out by CRISPR/Cas9 gene editing technology,and the gene knockout was identified as successful using PCR amplification,sequencing and real-time fluorescence quantitative PCR.(2)Effect of aprt gene knockout on the biological properties of CHO cells.Biological characterization was performed to verify whether the defective CHO cell lines could undergo normal growth and passages by observing cell morphology and growth status.The proliferation of defective cells was detected by CCK-8 method and doubling time calculation formula,respectively;the apoptosis of defective cells was detected by flow cytometry.(3)Construction of eukaryotic expression vector of recombinant protein.Based on the eukaryotic expression vector expressing the reporter gene enhanced green fluorescence protein(e GFP)constructed in our laboratory,an aprt expression cassette containing SV40 promoter attenuated or(and)aprt gene start codon mutation was inserted upstream of the e GFP expression cassette,which was named attenuated expression vector and mutant expression vector respectively.The corresponding eukaryotic expression vector of the target protein is to replace e GFP with human vitronectin(VN)or adalimumab(ADM)gene sequence,respectively.The constructed eukaryotic expression vectors were transfected into wild-type and APRT-deficient CHO cells respectively,and the stably transfected cell pool was screened.(4)Recombinant protein expression analysis.The recombinant CHO-e GFP cell pool was obtained by transfection screening and subcultured for 60 generations.The mean fluorescence intensity(MFI)of e GFP expression was detected by flow cytometry,and the expression stability of e GFP in different experimental groups was compared.The recombinant CHO-VN and CHO-ADM cell pools were obtained by transfection screening,and the expression levels of recombinant proteins in different experimental groups were compared by Western blot.Results(1)The aprt gene was successfully knocked out in CHO cells using PCR amplification,sequencing and real-time fluorescence quantitative PCR to identify the successful aprt gene knockout(genomic DNA fragment deletion of 141 bp).(2)The obtained APRT-deficient CHO cell lines were consistent with wild-type CHO cells in cell proliferation and apoptosis assays,and there was no significant difference in the ploidy time between the two groups(WT-CHO: 17.69 ± 0.3 h,APRT-KO:17.99 ± 0.2 h)(P > 0.05).the biological properties of APRT-deficient CHO cell lines were not significantly different from those of wild-type CHO cells did not differ significantly.(3)Transient expression results showed that the expression of e GFP was significantly higher in the recombinant APRT-deficient CHO cell line compared with wild-type CHO cells,and the expression was increased by 42% ± 6% and 56% ± 9% by transfection of the weakened expression vector and mutant expression vector,respectively.(4)The results of long-term subculture showed that the expression of EGFP protein could be significantly increased by transfecting defective cells with weakened vector or mutant vector under pressure or no pressure of pyricularin(P < 0.05).Among them,the expression of EGFP in defective cells transfected with mutant vector was the highest,and its average fluorescence intensity was 2.02 times that of wild-type cells transfected with control vector.Moreover,the expression of EGFP in two groups of recombinant CHO cells transfected with mutant vector was relatively stable,and the long-term expression maintenance rate of EGFP protein was higher than 70%.(5)The expressions of VN protein and ADM protein in defective CHO cells were higher than those in wild-type CHO cells.Conclusion(1)APRT-deficient CHO cell lines were established by CRISPR/Cas9 gene editing technology,and there were no significant differences between the defective cell lines and wild-type CHO cells in terms of biological properties.(2)The established APRT-deficient gene amplification system was able to significantly improve the recombinant protein expression level and expression stability,providing an effective genetic engineering strategy for the eventual establishment of an efficient and stable CHO cell expression system.