Expression of recombinant human GM2-activator protein in insect cells: purification and characterization by mass spectrometry.
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).
Wendeler
Michaela
M
Kekulé-Institut für Organische Chemie und Biochemie, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany.
Lemm
Thorsten
T
Weisgerber
Judith
J
Hoernschemeyer
Joerg
J
Bartelsen
Oliver
O
Schepers
Ute
U
Sandhoff
Konrad
K
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002412:Cations; D002460:Cell Line; D002461:Cell Line, Transformed; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D018076:DNA, Complementary / Q000378:metabolism; D049289:G(M2) Activator Protein; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D007313:Insecta; D013058:Mass Spectrometry; D008957:Models, Genetic; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D002412:Cations; D018076:DNA, Complementary; D049289:G(M2) Activator Protein; D009844:Oligosaccharides; D011506:Proteins; D011994:Recombinant Proteins