Knockout of caspase-7 gene improves the expression of recombinant protein in CHO cell line through the cell cycle arrest in G2/M phase

BACKGROUND: Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. RESULTS: Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. CONCLUSIONS: These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.

Weakening resistance marker for establishing a process of screening high-producing CHO cell lines / 中国药科大学学报

To optimize Chinese hamster ovary (CHO)expression system and establish a process of screening CHO cell lines with high productivity;neomycin-phosphotransferase (NPT)expressed by the resistance marker gene on the expression vector was mutated with amino acid D at 261 changed to G.After selection by culturing with G418;the survival rate of CHO cells bearing mutant-NPT was significantly lower than that of the cells bear-ing wide type NPT.An enhanced green fluorescent protein (EGFP)was genetically linked to the N terminus of the IgG1 Fc fragment part to generate an EGFP-Fc fusion protein regarded as a report gene;which verified that the resistance of mutant-NPT to G418 was weakened.By comparing fluorescence assay of EGFP intensity in stable transfections after selection with the same concentration of G418 for 3 weeks;mutant-selected pools expressed more exogenous protein than the WT-selected pools.Therefore;the ratio of high producers in a transfected cell population greatly increased.

Stable expression of targeting complement inhibitor CR2-CD59 in Chinese hamster ovary cells / 第三军医大学学报

Objective To obtain Chinese hamster ovary (CHO) cell lines that stably express a targeting complement inhibitor CR2-CD59.Methods The recombinant plasmid PEE14.1-CR2-CD59 was constru-cted by cloning the DNA fragment CR2-CD59 into plasmid PEE14.1,and the obtained plasmid was transfected into CHO cells by FuGENE 6.The clones with stable high expression of target fragment were selected by methionine sulfoximine (MSX),the expression of CR2-CD59 was analyzed by ELISA,SDS-PAGE and Western blotting analysis.Results Several stable expression clones were obtained,and CR2-CD59 was highly expressed in the secret form in CHO cells.SDS-PAGE analysis showed that the molecular weight of the recombined protein CR2-CD59 was consistent with the predicted one.ELISA and Western blotting results revealed that the CR2-CD59 could react with both anti-human CR2 and anti-human CD59 polyclonal antibodies.Compared with serum-containing medium,the protein was highly expressed in serum-free medium (P