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Biotechnology and Applied Biochemistry (2000) 32, (167–172) (Printed in Great Britain)

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Efficiency of promoter and cell line in high-level expression of erythropoietin

Jang Hyeon Park*¹, Chun Kim†, Won Bae Kim*, Young Kook Kim‡, Se Young Lee§ and Jai Myung Yang†2

*Research Laboratories of Dona-A Pharm Co., Ltd., Yongin-Si, Kyunggi-Do 449-900, South Korea, †Mapo-gu, Shinsu-dong, 1, Department of Life Science, Sogang University, Seoul 121-742, South Korea, ‡Korea Research Institute of Bioscience and Biotechnology, Taejon 305-600, South Korea, and §Graduate School of Biotechnology, Korea University, Seoul 136-701, South Korea

¹ Present address: Gen Tec Bio Co., Ltd., Seoul 135-260, South Korea.

² To whom correspondence should be addressed (e-mail jaimyang@ccs.sogang.ac.kr).

Efficiency of viral promoters and various cell lines in directing high-level expression of human erythropoietin (Epo) was investigated. To investigate the effects of various viral promoters and cell lines on the Epo expression level, genomic Epo with the 5´ and 3´ untranslated regions (UTRs) deleted was cloned next to the simian virus 40 early promoter, cytomegalovirus early promoter or SRa promoter. These expression vectors were transfected into COS-7, BHK-21 and Chinese hamster ovary (CHO)/dhfr^- cells, respectively. The COS-7 cells transfected with the vector containing the SRa promoter showed the highest expression level ( 103 IU/ml) at 72 h post-transfection. For the development of Epo-producing stable cell lines, BHK-21 and CHO/dhfr^- cells transfected with the 5´,3´-UTR-deleted genomic Epo under the control of the SRa promoter were cultured with media containing zeocin. Several clones of zeocin-resistant BHK-21 and CHO/dhfr^- cells were cultured in the presence of methotrexate (MTX). A BHK-21 clone selected in the presence of 500 nM MTX expressed and secreted 490 IU/ml Epo into the medium. A CHO/dhfr^- clone selected in the presence of 20 nM MTX expressed and secreted 45 IU/ml Epo into the medium. Southern-blot analysis indicated that enhancement of Epo expression in the MTX-resistant stable cells might be related to the amplification of gene copy number.

Received 4 August 2000/4 September 2000; accepted 5 September 2000

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