Multigene Metabolic Engineering Of HEK293 Cells Based On Biotin-inducible Expression System

In biopharmaceutical industry, mammalian cell expression systems, including CHO, HEK293 and BHK cells, are preferred for the production of large, complex proteins requiring post-translation modification. Among these, HEK293 cell line, transformed from human embryonic kidney, with the exclusive abilities of propeptide cleavage, gamma-carboxylation and human-type glycosylation, is increasingly important in the production of biotech drugs. Like other mammalian cell expression systems, HEK293 cells in large-scale culture are confronted with the problem of decreased production efficiency resulted from apoptosis and excessive cell proliferation, affecting the productivity and quality of recombinant proteins.Metabolic engineering approaches incorporating antiapoptosis engineering and controlled proliferation technology, combined with multicistronic expression and inducible gene expression, are used in directed genetic engineering of mammalian cells, endowing them the in vitro characteristics of antiapoptosis and controlled proliferation. In this study, HEK293 cells were modified to constitutively overexpress anti-apoptotic gene E1B-19K and inducibly express cell-cycle regulating gene p27^Kip1. By multigene metabolic engineering based on biotin-inducible expression system, HEK293 cells were improved in the abilities of antiapoptosis and proliferation control.E1B-19K gene was obtained by overlapping PCR and ligated into pcDNA3.1(+) to produce E1B-19K-expressing vector, pc-E1B. Compared to HEK293 cells transfected with blank vector, the cells stably transfected with pc-E1B were investigated in the growth under culture conditions with low glucose concentration, reduced serum or deprived glutamine. Under the above conditions, the expression of E1B-19K reduced apoptosis by 60-80%. In normal culture condition, E1B-19K- expressing cells showed higher viabilities and delayed decline phase by 2 days. No significant influences on Qglc, Qlac and Qgln were observed after the expression of E1B-19K.Tet-responsive promoter PhCMV*-1 was amplified from pTRE vector and ligated into pc-Hy to construct Dox-inducible expression vector pc-Hy-star. P27^Kip1 cDNA was cloned from pCMV-SPORT6-p27 and inserted downstream of PhCMV*-1 of pc-Hy-star. The resulting vector could express p27^Kip1 responsive to Dox and was named pc-Hy-star-p27. HEK293 cells were co-transfected with the vectors pTet-on and pc-Hy-star-p27 and cell growth and metabolism were assessed with cell-cycle distribution and viable cell density, Qglc, Qlac and Qgln as indexes, respectively. The results showed that the expression of p27^Kip1 led to slowed cell growth, increased percentage of G1 cells, reduced consumption of glucose and production of lactate.BS-BirA cloned from BS 168 genome and VP4 cut from pc-Hy-GVP4 were assembled by overlapping PCR to obtain BS-BirA-VP4, a fusion transactivator, and BS-BirA-VP4 was inserted downstream of PCMV of pcDNA3.1(+) to produce the regulatory vector pc-BS-BV. PCMV in pc-Hy-E was replaced by the minimal CMV promoter PminCMV from pTRE to obtain pc-Hy-minE and then synthetic four tandem repeats of BSOB were inserted upstream of pc-Hy-minE to produce the response vector pc-Hy-4BSOB-minE. Thereby, a novel gene regulation system BS-Biotin-On, based on BS-BirA and responsive to biotin, was established. In this system, BS-BirA-VP4 bound specifically to BSOB in the presence of biotin and activated the PminCMV-drived transcription of intrest gene. The regulatory vector pc-BS-BV and EGFP-expressing response vector pc-Hy-4BSOB-minE were transfected into HEK293 cells and the effect of BS-Biotin-On system was investigated by measuring EGFP relative fluorescence tensity. The results validated this system in its rapid response, low leaky expression and high inducing rate.E1B-19K and BS-BirA-VP4 were respectively inserted downstream of PEF-1αand PCMV of pBudCE4.1 vetor to construst pBud-E1B-BS-BV vector for simultaneous and independent expression of these two genes. P27^Kip1 from pc-Hy-star-p27 and IRES-EGFP from pIRES2-EGPF were sequentially ligated into pc-Hy-4BSOB-minE vector with EGFP gene removed. The resulting vector pc-Hy-4BSOB-minp27-IE could mediate biotin- regulated expression of p27^Kip1 coupled with EGFP. The above two vectors were transfected into HEK293 cells and single-cell cloning was carried out by FACS-sorting and limiting dilution. HEK-EPIE-18 cell line constitutively expressing E1B-19K and biotin-regulated expressing p27^Kip1 was obtained. In the presence of biotin, percentage of G1 cells in the late culture phase of HEK-EPIE-18 increased from 47.88 % to 66.92 % and apoptosis in spent medium was reduced by 47-54% compared with control blank-transfected HEK293 (HEK-CT). These results suggested that the constructed HEK-EPIE-18 cell line possessed the in vitro culture characteristics of antiapoptosis and proliferation control.In order to validate the effect of multigene metabolic engineering on exogenous protein expression, rhTNFR-Fc-expressing vector pc-TNFR-Fc was transfected into HEK-CT and HEK-EPIE cells and stably-transfected clones were compared in Qp and cumulative level of rhTNFR-Fc in serum-free suspension culture. In the absence of biotin, compared with the pc-TNFR-Fc-transfected HEK-CT cells, transfected HEK-EPIE-18 cells showed indistictive increase in Qp, obvious enhancement in viable cell density and cell viability, with an increase of cumulative yield of rhTNFR-Fc by 56.8%. In the presence of biotin, pc-TNFR-Fc-transfected HEK-EPIE-18 cells showed lowed proliferate rate, high viability above 90%, doubled Qp of rhTNFR-Fc, and increased cumulative yield of rhTNFR-Fc by 1.1 folds compared with transfected HEK-CT cells.To sum up, our study indicate multigene metabolic engineering strategy, integrating constitutive overexpression of antiapoptotic E1B-19K with biotin- inducible expression of cell-cycle regulatory p27^Kip1, is highly effective for mammalian cells in improving in vitro growth characteristics and enhancing the efficiency of product expression.