Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris, purification, isotopic labeling, and biophysical characterization.
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.
Wendeler
Michaela
M
Kekulé-Institut für Organische Chemie und Biochemie, Gerhard-Domagk-Strasse 1, Bonn D-53121, Germany.
Hoernschemeyer
Joerg
J
John
Michael
M
Werth
Norbert
N
Schoeniger
Maike
M
Lemm
Thorsten
T
Hartmann
Rudolf
R
Kessler
Horst
H
Sandhoff
Konrad
K
D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D049289:G(M2) Activator Protein; D005678:G(M2) Ganglioside / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008055:Lipids / Q000032:analysis; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D000432:Methanol / Q000494:pharmacology; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010843:Pichia / Q000235:genetics*; D011134:Polysaccharides / Q000737:chemistry; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance; D014170:Transformation, Genetic; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D018076:DNA, Complementary; D049289:G(M2) Activator Protein; D008055:Lipids; D008081:Liposomes; D009587:Nitrogen Isotopes; D011134:Polysaccharides; D011506:Proteins; D011994:Recombinant Proteins; D005678:G(M2) Ganglioside; D001619:beta-N-Acetylhexosaminidases; D000432:Methanol