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.
Over-expression of a functionally active human GM2-activator protein in Escherichia coli
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H Klima, A Klein, G van Echten, G Schwarzmann, K Suzuki, K Sandhoff; Over-expression of a functionally active human GM2-activator protein in Escherichia coli. Biochem J 1 June 1993; 292 (2): 571–576. doi: https://doi.org/10.1042/bj2920571
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