| Expression of recombinant human GM2-activator protein in insect cells: purification and characterization by mass spectrometry. | |
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MedLine Citation:
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PMID: 12597885 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The GM2-activator protein (GM2AP) is a small non-enzymatic cofactor assisting the enzyme beta-hexosaminidase A in the lysosomal degradation of ganglioside GM2. Mutations in the gene encoding this glycoprotein lead to a fatal neurological disorder, the AB variant of GM2-gangliosidoses. In this paper, we describe the overexpression of GM2AP in Sf21 cells using both the baculovirus expression vector system (BEVS) and a non-lytic, plasmid-based insect cell expression system (InsectSelect). For the BEVS, the cDNA encoding human GM2AP-preproprotein was cloned in the expression vector pAcMP3. The recombinant virus generated by cotransfection with linearized baculovirus DNA was used to infect Sf21 cells. For the non-lytic expression system, the cDNA of GM2AP was inserted into the vector pIZ/V5-His, which was used for the constitutive expression in stably transformed Sf21 cells. As it was shown by immunoblot analysis of the cell culture supernatant, in both expression systems the GM2AP precursor protein was efficiently secreted into the medium. Following expression in the BEVS, the GM2AP was purified by sequential chromatography on Ni-NTA-agarose and Con A-Sepharose, resulting in a yield of up to 9 mg purified protein from 1L of cell culture supernatant. Following expression in stably transformed insect cells, the secreted protein was first concentrated by cation-exchange and purified by metal-ion affinity chromatography, with a yield of 0.1 mg/L cell culture supernatant. The biological activity of the recombinant protein was demonstrated by its ability to stimulate the hexosaminidase A-catalyzed degradation of ganglioside GM2, and the homogeneity and glycosylation were assessed by ESI-TOF mass spectrometry. While the protein expression in the BEVS led to partly glycosylated and partly non-glycosylated protein, the stably transformed cells produced only glycosylated protein. In both expression systems, the glycosylation was found to be identical and corresponded to the structure (GlcNAc)(2)Fuc(Man)(3). |
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Authors:
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Michaela Wendeler; Thorsten Lemm; Judith Weisgerber; Joerg Hoernschemeyer; Oliver Bartelsen; Ute Schepers; Konrad Sandhoff |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Protein expression and purification Volume: 27 ISSN: 1046-5928 ISO Abbreviation: Protein Expr. Purif. Publication Date: 2003 Feb |
Date Detail:
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Created Date: 2003-02-24 Completed Date: 2003-10-09 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 9101496 Medline TA: Protein Expr Purif Country: United States |
Other Details:
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Languages: eng Pagination: 259-66 Citation Subset: IM |
Copyright Information:
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Copyright 2002 Elsevier Science (USA) |
Affiliation:
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Kekulé-Institut für Organische Chemie und Biochemie, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals Baculoviridae / metabolism Cations Cell Line Cell Line, Transformed Chromatography Chromatography, Ion Exchange DNA, Complementary / metabolism G(M2) Activator Protein Genetic Vectors Glycosylation Humans Immunoblotting Insects Mass Spectrometry Models, Genetic Mutation Oligosaccharides / chemistry Proteins / chemistry*, isolation & purification, metabolism* Recombinant Proteins / chemistry, metabolism Transfection |
| Chemical | |
Reg. No./Substance:
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0/Cations; 0/DNA, Complementary; 0/G(M2) Activator Protein; 0/Oligosaccharides; 0/Proteins; 0/Recombinant Proteins |
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