H. Klima et al., OVER-EXPRESSION OF A FUNCTIONALLY ACTIVE HUMAN G(M2)-ACTIVATOR PROTEIN IN ESCHERICHIA-COLI, Biochemical journal, 292, 1993, pp. 571-576
The cDNA of the human G(M2)-activator protein was cloned into the expr
ession vector pHX17. The plasmid encodes a fusion protein with a hexah
istidine tail and a Factor Xa cleavage site at its N-terminus. The rec
ombinant protein was purified from cell homogenates under denaturing c
onditions by metal-ion affinity chromatography in a single step and th
en was refolded. The hexahistidine tail could be removed when desired
by digestion with Factor Xa. In a functional assay, the G(M2)-activato
r thus generated from Escherichia coli and renatured, with or without
the hexahistidine tail, was as active as the native G(M2)-activator pr
otein that was purified from human tissue. When added to the culture m
edium, the recombinant carbohydrate-free G(M2)-activator, carrying the
hexahistidine tail, could be taken up efficiently and restored the de
gradation of ganglioside G(M2) to normal rates in mutant fibroblasts w
ith the AB variant of G(M2)-gangliosidosis, which is characterized by
a genetic defect in the G(M2)-activator protein. The prokaryotic expre
ssion system is useful for producing milligram quantities of a pure an
d functionally active G(M2)-activator.