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  High-Level Secretion of Biologically Active Recombinant Human Macrophage Inflammatory Protein-1α by the Methylotrophic Yeast Pichia pastoris

Maeda, Y., Kuroki, R., Suzuki, H., & Reiländer, H. (2000). High-Level Secretion of Biologically Active Recombinant Human Macrophage Inflammatory Protein-1α by the Methylotrophic Yeast Pichia pastoris. Protein Expression and Purification, 18(1), 56-63. doi:10.1006/prep.1999.1156.

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 Creators:
Maeda, Yoshitake1, Author              
Kuroki, Ryota2, Author
Suzuki, Hidefumi3, Author
Reiländer, Helmut1, Author              
Affiliations:
1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              
2Central Laboratories for Key Technology, KIRIN Brewery Co., LTD. 1-13-5 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan, ou_persistent22              
3Pharmaceutical Research Laboratory, KIRIN Brewery CO., LTD. 3 Miyahara-Cho, Takasaki, Gunma, 370 1295, ou_persistent22              

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 Abstract: The human CC chemokine macrophage inflammatory protein-1α (MIP-1α) was produced at a high level in Pichia pastoris under transcriptional control of the highly inducible alcohol oxidase 1 promoter. To ensure proper folding and secretion of the recombinant polypeptide, the MIP-1α gene had been fused to the Saccharomyces cerevisiae α-factor prepropeptide. As was revealed by analysis of the cell culture supernatant of recombinant Pichia pastoris, MIP-1α was efficiently secreted. Immunoblot analysis of secreted proteins from recombinant clones using a polyclonal antibody directed against MIP-1α revealed an apparent molecular mass of 8 kDa for the recombinant polypeptide. Up to 70 mg of MIP-1α was purified from 1 liter of yeast culture supernatant by a single chromatography step. Biological activity of recombinant MIP-1α was shown in a chemotaxis assay. Here, the polypeptide specifically induced migration of U937 cells expressing the CCR1 (MIP-1α receptor). Also, in competition binding assays the recombinant MIP-1α displayed high affinity binding.

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Language(s): eng - English
 Dates: 1999-09-061999-07-082002-05-252000-02-01
 Publication Status: Published in print
 Pages: 8
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1006/prep.1999.1156
 Degree: -

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Title: Protein Expression and Purification
Source Genre: Journal
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Publ. Info: San Diego : Academic Press
Pages: - Volume / Issue: 18 (1) Sequence Number: - Start / End Page: 56 - 63 Identifier: ISSN: 1046-5928
CoNE: https://pure.mpg.de/cone/journals/resource/954922650158