Isolation and expression of a full-length cDNA encoding the human G M2 activator protein

Abstract

We report the construction of a cDNA clone encoding a functional G M2-activator protein. The sequence of the complete 5′ end of the coding region was determined by direct nucleotide sequencing of a fragment generated by multiple RACE PCR procedures from Hela cell cDNA. Specific oligonucleotides were synthesized from these data which allowed us to produce a PCR fragment that contained the complete coding sequence of the protein. This was then cloned into a mammalian expression vector. The ability of purified hexosaminidase A (β-N-acetylhexosaminidase, EC 3.2.1.52) to hydrolyse labeled G M2 ganglioside was enhanced 10-fold more by the addition in the assay mix of lysate from transfected COS-1 cells than by the addition of identical amounts of lysate from mock transfected cells. Direct sequencing of PCR fragments from two sources also identified three polymorphisms.