Linked Life Data

14766311

Source:http://linkedlifedata.com/resource/pubmed/id/14766311

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2004-2-9
pubmed:abstractText
The GM2-activator protein (GM2AP) belongs to a group of five small, nonenzymatic proteins that are essential cofactors for the degradation of glycosphingolipids in the lysosome. It mediates the interaction between the water-soluble enzyme beta-hexosaminidase A and its membrane-embedded substrate, ganglioside GM2, at the lipid-water interphase. Inherited defects in the gene encoding this glycoprotein cause a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. With the aim to establish a convenient eukaryotic system that allows the efficient production of functionally folded, glycosylated GM2AP and offers the potential of cost-efficient isotopic labeling for structural studies by NMR spectroscopy, we established the expression of recombinant GM2AP in the methylotrophic yeast Pichia pastoris. For the construction of expression plasmids, either the full cDNA encoding human GM2AP preproprotein was cloned in the expression vector pPIC3.5K, or the cDNA encoding only the mature form of GM2AP was inserted in the vector pPIC9K under control of the alcohol oxidase 1 promoter. Both plasmids led to the successful secretory expression of active, glycosylated GM2AP, which could easily be purified by Ni-NTA chromatography due to the hexahistidine tag introduced at the C-terminus. Remarkably, the expression of this membrane-active protein in P. pastoris was accompanied by two peculiarities which were not encountered in other expression systems for GM2AP: First, a significant fraction of the secreted protein existed in the form of aggregates, and second, considerable amounts of noncovalently bound lipids were associated with the recombinant protein. A three-step purification scheme was therefore devised consisting of Ni-NTA, reversed phase, and gel filtration chromatography, which finally yielded 10-12 mg of purified, monomeric GM2AP per liter of expression supernatant. MALDI- and ESI-TOF mass spectrometry were employed to assess the processing, homogeneity, and glycosylation pattern of the recombinant protein. Surface plasmon resonance spectroscopy allowed the interaction of GM2AP with immobilized liposomes to be studied. A modified version of FM22 minimal medium was then used in the cost-effective (15)N-labeling of GM2AP to assess its amenability for the structural investigation by NMR spectroscopy. Initial (15)N,(1)H-HSQC experiments show a well-folded protein and provide evidence for extensive conformational exchange processes within the molecule.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
1046-5928
pubmed:author
pubmed:issnType
Print
pubmed:volume
34
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
147-57
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed-meshheading:14766311-Chromatography, pubmed-meshheading:14766311-Chromatography, Gel, pubmed-meshheading:14766311-Chromatography, Thin Layer, pubmed-meshheading:14766311-DNA, Complementary, pubmed-meshheading:14766311-Electrophoresis, Polyacrylamide Gel, pubmed-meshheading:14766311-G(M2) Activator Protein, pubmed-meshheading:14766311-G(M2) Ganglioside, pubmed-meshheading:14766311-Gene Expression, pubmed-meshheading:14766311-Genetic Vectors, pubmed-meshheading:14766311-Glycosylation, pubmed-meshheading:14766311-Humans, pubmed-meshheading:14766311-Lipids, pubmed-meshheading:14766311-Liposomes, pubmed-meshheading:14766311-Methanol, pubmed-meshheading:14766311-Nitrogen Isotopes, pubmed-meshheading:14766311-Nuclear Magnetic Resonance, Biomolecular, pubmed-meshheading:14766311-Pichia, pubmed-meshheading:14766311-Polysaccharides, pubmed-meshheading:14766311-Protein Biosynthesis, pubmed-meshheading:14766311-Proteins, pubmed-meshheading:14766311-Recombinant Proteins, pubmed-meshheading:14766311-Spectrometry, Mass, Electrospray Ionization, pubmed-meshheading:14766311-Spectrometry, Mass, Matrix-Assisted Laser..., pubmed-meshheading:14766311-Surface Plasmon Resonance, pubmed-meshheading:14766311-Transformation, Genetic, pubmed-meshheading:14766311-beta-N-Acetylhexosaminidases
pubmed:year
2004
pubmed:articleTitle
Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris, purification, isotopic labeling, and biophysical characterization.
pubmed:affiliation
Kekulé-Institut für Organische Chemie und Biochemie, Gerhard-Domagk-Strasse 1, Bonn D-53121, Germany.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't