High Level Secretion by Saccharomyces cerevisiae of Human Apolipoprotein E as a Fusion to Rhizomucor Rennin
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概要
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As the first step for production of human apolipoprotein E (hApoE) in Saccharomyces cerevisiae, the hApoE cDNA was cloned in Escherichia coli, on the basis of the nucleotide sequence reported previously. When the hApoE cDNA including its pre-sequence-encoding region was expressed under the control of the GAL7 promoter, no protein immunoreactive with anti-hApoE antibody was detected either in the culture medium or inside the cells. For efficient production and secretion of hApoE in S. cerevisiae, the mature hApoE-encoding region was fused to the prepro-sequence region of Rhizomucor rennin (MPR) and to the whole MPR gene including its prepro- and mature-MPR regions. When the fusion gene consisting of the prepro-sequence-encoding region and hApoE regions was expressed in S. cerevisiae, no protein reactive with the anti-hApoE antibody was detected in any fraction of the yeast cells, probably due to rapid degradation of the hApoE protein by yeast proteases. On the othe hand, when hApoE was expressed as a fusion to the whole MPR protein, a considerable amount of the fused protein was secreted into the medium. The prepro-sequence of MPR was correctly processed from the fused protein in the medium by autocatalytic activity of MPR and by a protease(s) of the host cell. Integration of the fusion gene into the chromosome at a copy number of eight led to secretion of the fused protein in a larger amount than the case when the fusion gene was carried on a 2-μm plasmid with its copy number of a few hundreds, because the 2-μm derived plasmid containing the fusion gene was very unstable in the yeast cells. The secretion level was also improved by changing the culture conditions. A maximum yield of hApoE part in the secreted fused protein was estimated to be 23. 7mg per liter and the amount of the fused protein was calculated to be 53. 0mg per liter.
- 社団法人日本農芸化学会の論文
- 1995-03-23
著者
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HORINOUCHI Sueharu
Department of Biotechnology, University of Tokyo
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Nomura Nobuhiko
Department Of Biotechnology The University Of Tokyo
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BEPPU Teruhiko
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tok
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Yamada Hisashi
Fermentation Development Laboratories Fermentation Development Laboratories Fujisawa Pharmaceutical
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Horinouchi Sueharu
Department Of Agricultural Chemistry Faculty Of Agriculture The University Of Tokyo
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Horinouchi Sueharu
Dep. Of Biotechnology Graduate School Of Agriculture And Life Sciences Univ. Of Tokyo
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Yamada H
Fermentation Development Laboratories Fermentation Development Laboratories Fujisawa Pharmaceutical
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Beppu T
Department Of Applied Biological Science Nihon University
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Yamada Hisashi
Research Laboratories, Toyama Chemical Co., Ltd.
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Matsubara Nobuyuki
Research Laboratories, Toyama Chemical Co., Ltd.
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Matsubara N
Snow Brand Milk Products Co. Ltd. Saitama Jpn
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Matsubara Nobuyuki
Research Laboratories Toyama Chemical Co. Ltd.
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Beppu Teruhiko
Department Of Agricultural Chemistry Faculty Of Agriculture The University Of Tokyo
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