Over-expression of a functionally active human GM2-activator protein in Escherichia coli.
The cDNA of the human GM2-activator protein was cloned into the expression vector pHX17. The plasmid encodes a fusion protein with a hexahistidine tail and a Factor Xa cleavage site at its N-terminus. The recombinant protein was purified from cell homogenates under denaturing conditions by metal-ion affinity chromatography in a single step and then was refolded. The hexahistidine tail could be removed when desired by digestion with Factor Xa. In a functional assay, the GM2-activator thus generated from Escherichia coli and renatured, with or without the hexahistidine tail, was as active as the native GM2-activator protein that was purified from human tissue. When added to the culture medium, the recombinant carbohydrate-free GM2-activator, carrying the hexahistidine tail, could be taken up efficiently and restored the degradation of ganglioside GM2 to normal rates in mutant fibroblasts with the AB variant of GM2-gangliosidosis, which is characterized by a genetic defect in the GM2-activator protein. The prokaryotic expression system is useful for producing milligram quantities of a pure and functionally active GM2-activator.
Institut für Organische Chemie und Biochemie, Bonn, Germany.
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Klima
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Klein
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van Echten
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Schwarzmann
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Suzuki
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Sandhoff
D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli; D005347:Fibroblasts / Q000378:metabolism; D049289:G(M2) Activator Protein; D005678:G(M2) Ganglioside; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D049289:G(M2) Activator Protein; D011506:Proteins; D011994:Recombinant Proteins; D005678:G(M2) Ganglioside; D004247:DNA