OVER-EXPRESSION OF A FUNCTIONALLY ACTIVE HUMAN G(M2)-ACTIVATOR PROTEIN IN ESCHERICHIA-COLI

The cDNA of the human G(M2)-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 G(M2)-activator thus generated from Escherichia coli and renatured, with or without the hexahistidine tail, was as active as the native G(M2)-activator protein that was purified from human tissue. When added to the culture medium, the recombinant carbohydrate-free G(M2)-activator, carrying the hexahistidine tail, could be taken up efficiently and restored the degradation of ganglioside G(M2) to normal rates in mutant fibroblasts with the AB variant of G(M2)-gangliosidosis, which is characterized by a genetic defect in the G(M2)-activator protein. The prokaryotic expression system is useful for producing milligram quantities of a pure and functionally active G(M2)-activator.