Simultaneous recovery of dual pathways for ammonia metabolism do not improve further detoxification of ammonia in HepG2 cells

BACKGROUND:Key enzyme deficiency in the dual-pathway of ammonia metabolism leads to low detoxification capacity of HepG2 cells.Previously,we established a HepG2/AFhGS cell line with overexpression of human glutamine synthetase(hGS) in pathway 1 and a HepG2/(hArgI+hOTC)4 cell line with overexpression of human arginase I(hArgI) and human ornithine transcarbamylase(hOTC) in pathway 2.The present study aimed to investigate whether simultaneous recovery of the two pathways contributes to the further improvement of ammonia detoxification in HepG2 cells.METHODS:We adopted a recombinant retrovirus carrying the hGS gene to infect HepG2/(hArgI+hOTC)4 cells and selected a new recombinant HepG2 cell line.The capacities of ammonia tolerance and detoxification in cells were detected by biochemical methods.Cell cycle PCR chip was used to assess the changes of gene expression.RESULTS:Introducing hGS into HepG2/(hArgI+hOTC)4 cells did not lead to hGS overexpression,but inhibited hArgI expression.The levels of synthetic glutamine and urea in HepG2/(hArgI+hOTC+AFhGS)1 cells were significantly lower than those in HepG2/(hArgI+hOTC)4 cells when cultured in the medium with 10 and 15 mmol/L glutamate(Glu) and with 60 and 180 mmol/L NH 4 Cl,respectively.In addition,the comparison of different cell growth showed that HepG2/AFhGS cells significantly lagged behind the other cells by the 5th and 7th day,indicating that introduction of hGS impedes HepG2 cell proliferation.Analysis of the mechanism suggested that the decreased expression of BCL2 played an important role.CONCLUSIONS:This study demonstrated that the recovery of two ammonia metabolic pathways in HepG2 cells is not helpful in increasing ammonia metabolism.The reinforcement of the pathway of urea metabolism is more important and valuable in improving the ammonia metabolism capacity in HepG2 cells.

High expression level of soluble SARS spike protein mediated by adenovirus in HEK293 cells

AIM： To develop a highly efficacious method for preparation of soluble SAPS S-protein using adenovirus vector to meet the requirement for S-protein investigation. METHODS： The human adenovirus vector was used to express the soluble S-protein （corresponding to 1-1190 amino acids） fused with Myc/His tag using codon-optimized gene construct in HEK239 cells. The recombinant adenovirus bearing S-protein gene was generated by ligation method. The expressed S-protein with Myc/His tag was purified from culture medium with Ni-NTA agarose beads followed by dialysis. The S-protein was detected by Western blot and its biologic activity was analyzed by binding to Vero cells. RESULTS： Under the conditions of infection dose （MOI of 50） and expression time （48 h）, the high-level expression of S-protein was obtained. The expression level was determined to be approximately 75 μg/106 cells after purification. Purified soluble S-protein was readily detected by Western blot with anti-Myc antibody and showed the ability to bind to surface of Vero cells, demonstrating that the soluble S-protein could remain the biologic activity in the native molecule. CONCLUSION： The high-level expression of S-protein in HEK293 cells mediated by adenovirus can be achieved under the optimized expression conditions. The proteins possess the biologic activity, which lays a foundation for further investigation of S-protein biological function.