Overexpression Of ATF3 And CK2? Enhancing The Recombinant Protein Production In CHO Cells And The Mechanism Study

Background and purpose:Chinese hamster ovary(CHO)cells are one of the most popular host cells for the production of recombinant proteins,monoclonal antibodies and biomacromolecule drugs,especially suitable for those need complex posttranslational modifications,such as all kinds of full-length monoclonal antibodies.However,compared with the expression systems of E.coli and yeast,the cost of mammalian cells such as CHO is much higher.The product is expensive and unaffordable for many patients.Therefore,it is of great significance to improve the growth performance and productivity of exogenous protein of CHO engineering cell line to decrease cost and improve market accessibility.In recent years,with the emergence of new techniques such as the next generation sequencing,the high-efficiency gene editing tools and other technologies,functional gene screening and gene editing of CHO cells become possible.In our lab,the knock-out of DNA methyltransferase 3A(DNMT3a)in CHO cells was carried out previously.On this basis,RNAseq showed that the transcription level of ATF3 gene was up-regulated by DNMT3 a deficiency,which may be related to the growth and metabolism alterations of CHO cells.On the other hand,clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats bind protein 9(CRISPR/cas9)was used to establish recombinant stale cell lines by inserting foreign gene into specific loci such as C12orf35 and HPRT in the genome of CHO cells.In this study,we selected two genes to study the effect of their overexpression in CHO cells,one is the above mentioned ATF3,the other is casein kinase ?(CK2),and both of them showed impact on cell activity in literatures.Methods:In order to investigate the effects of ATF3 and CK2? on the proliferation and exogenous protein expression in CHO cells,we first performed transient expression of ATF3 and CK2? in KCSG6,a cell line we constructed before harboring the red fluorescence protein(mCherry).After1?5 days of transfection,cell proliferation as well as m Cherry protein level were analyzed to evaluate the functions of ATF3 and CK2?.In order to study the functions of ATF3 and CK2? genes more rigorously,we used CRISPR/Cas9 mediated stable cell line construction technology,selected HPRT as the integration site,and constructed donor plasmids ATF3-Donor,CK2?-Donor and ATF3+CK2?-Donor.The stable pools after electroporation were obtained and enriched by neomycin pressure,then analyzed by 5'/3' junction PCR.After limiting-dilution,the monoclonal cells were identified and sequenced by 5'/3' junction PCR and out-out PCR with limited dilution method.The expression of ATF3,CK2?and ATF3+CK2? in the selected cell lines were further detected.A total of 3stable cell lines were obtained.In order to study the effect and mechanism of ATF3 and CK2?,the cell apoptosis and related signal pathway molecules were analyzed.Results:The transient transfection of ATF3 and CK2? showed that both ATF3 and CK2? had a trend of promoting cell proliferation and protein expression.By CRISPR/cas9 technology,we successfully insert ATF3,CK2?,and ATF3+CK2? into HPRT sites of KCSG6 cell line and finally obtained 3stable cell lines: ATF3-8,CK2?-3,AC-2.We observed the inserted gene was successfully expressed,which promoted cell proliferation,and CK2? can promoted the production of m Cherry.By comparing the cell cycle and apoptosis of these three cell lines with the blank KCSG cell,we found that ATF3+CK2? can inhibit the total apoptosis of cells,but the single ATF3 or CK2? has no such effects.All three genes improved the phosphorylation level of ERK pathway,which may contribute to their cell proliferation and protein expression functions.Furthermore,using the bisulfide PCR sequencing of CHO wild type and DNMT3 a deficient cells,we revealed that the methylation level of ATF3 gene promoter was regulated by DNMT3 a,which laid the foundation to understand the regulatory mechanism of ATF3.Overall,our study showed that the overexpression of ATF3 and CK2?gene can promote the cell proliferation and CK2? can promote the stimulate exogenous protein expression in CHO cells,which is of potential significance for cell line engineering in biopharmaceuticals development process.We also obtained the stable CHO cell lines with over expression of ATF3,CK2?,as well as ATF3+CK2? genes,which can be further studied in combination with the optimization of cell culture process,to constantly improve the level of biopharmaceuticals production.