MG-132 Improve the Production of TNFR-Fc Fusion Protein in CHO Cells

doi:10.13523/j.cb.20150901

China Biotechnology

2015, Vol. 35

Issue (9): 1-6 DOI: 10.13523/j.cb.20150901

MG-132 Improve the Production of TNFR-Fc Fusion Protein in CHO Cells

WEN Jia-ming¹, NIE Yan-feng^1,2, LIANG Han-zhang¹, HUANG Wen-xi¹, LEI Yun¹, XIE Qiu-ling¹, PEI Yun-lin², XIONG Sheng¹

1 College of Pharmacy, Institute of Biomedicine & National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou 510632, China;
2 Guangdong Marubi Biotechnology Company Limited, Guangzhou 510530, China

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Abstract

Objective: Try to improve the production and quality of TNFR-Fc fusion protein in CHO cells by inhibiting protein degradation. Methods: The stability and degradation pathways of TNFR-Fc in CHO cells were investigated by measuring the cell density and survival rate with additional lysosome inhibitor (Leupeptin) and ubiquitin-proteasome inhibitor (MG-132), respectively. The qualities of the total and extracellular TNFR-Fc expression were detected by Western blot and ELISA. Extracellular TNRF-Fc was purified by Protein A Affinity Chromato- graphy. The protein purity was analyzed by High Performance Liquid Chromatography (HPLC), while the enzyme activity of TNFR-Fc was assessed by measuring the inhibition level toward the cytotoxicity of L929 cell induced by TNFα. Results: TNFR-Fc was determined to be degraded via the ubiquitin-proteasome pathway;Adding 50 μmol/L MG-132 when cell culture, the production of total TNFR-Fc and dimer TNFR-Fc were improved 42.35% and 28.60%, respectively. Moreover, purified TNFR-Fc exhibited good bioactivity and the EC₅₀ is 2.86ng/ml. Conclusion: MG-132 adding when cell culture may become a competent strategy to improve the recombinant protein production of CHO cells expression system.

Key words： MG-132 TNFR-Fc degradation CHO cell Ubiquitination Improve production

Received: 11 March 2015 Published: 25 September 2015

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WEN Jia-ming, NIE Yan-feng, LIANG Han-zhang, HUANG Wen-xi, LEI Yun, XIE Qiu-ling, PEI Yun-lin, XIONG Sheng. MG-132 Improve the Production of TNFR-Fc Fusion Protein in CHO Cells. China Biotechnology, 2015, 35(9): 1-6.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20150901 OR https://manu60.magtech.com.cn/biotech/Y2015/V35/I9/1

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