| Purification and characterization of recombinant human granulocyte colony-stimulating factor (rhG-CSF) derivatives: KW-2228 and other derivatives. | |
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MedLine Citation:
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PMID: 9757559 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Various derivatives of recombinant human granulocyte colony-stimulating factor (rhG-CSF) have been overproduced in Escherichia coli with the strong, inducible trp promoter. A derivative designated as KW-2228 in which the amino acids were replaced at five positions showed more potent granulopoietic activity and stability than those of wild-type both in vitro and in vivo. The purification involved a sequential renaturation process and three-step chromatography. Refolding succeeded in very high yield using a urea system. The purity of KW-2228 was greater than 99% as measured by SDS-PAGE and HPLC analysis. According to circular dichroism and nuclear magnetic resonance spectroscopy, rhG-CSF and KW-2228 have very similar conformations. This suggests that the substitution of five amino acids does not appreciably change the conformation of hG-CSF. KW-2228 ([Ala1, Thr3, Tyr4, Arg5, and Ser17]-hG-CSF) and derivative A ([Ala1, Thr3, Tyr4, Arg5]-hG-CSF) are easily crystallized and they show similar in vitro activity. On the other hand, neither rhG-CSF nor derivative B ([Ser17]-hG-CSF) are crystallized under the same conditions. Thus, the four amino acid substitution (Ala1, Thr3, Tyr4, Arg5) of the N-terminal sequence may facilitate crystallization. The change of Cys17 to Ser may not influence the stability and activity of hG-CSF. |
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Authors:
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M Yamasaki; N Konishi; K Yamaguchi; S Itoh; Y Yokoo |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: Bioscience, biotechnology, and biochemistry Volume: 62 ISSN: 0916-8451 ISO Abbreviation: Biosci. Biotechnol. Biochem. Publication Date: 1998 Aug |
Date Detail:
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Created Date: 1998-10-29 Completed Date: 1998-10-29 Revised Date: 2004-11-17 |
Medline Journal Info:
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Nlm Unique ID: 9205717 Medline TA: Biosci Biotechnol Biochem Country: JAPAN |
Other Details:
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Languages: eng Pagination: 1528-34 Citation Subset: IM |
Affiliation:
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Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Japan. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acids
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analysis Animals Antineoplastic Agents / chemistry*, isolation & purification Biological Assay Chromatography, Gel Chromatography, High Pressure Liquid Circular Dichroism Colorimetry Crystallization Electrophoresis, Polyacrylamide Gel Escherichia coli / chemistry Granulocyte Colony-Stimulating Factor / chemistry*, isolation & purification Humans Magnetic Resonance Spectroscopy Mice Molecular Weight Protein Conformation Protein Folding Recombinant Proteins / chemistry*, isolation & purification Sequence Analysis |
| Chemical | |
Reg. No./Substance:
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0/Amino Acids; 0/Antineoplastic Agents; 0/Recombinant Proteins; 134088-74-7/nartograstim; 143011-72-7/Granulocyte Colony-Stimulating Factor |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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