Cloning and functional expression of a cDNA encoding coffee bean alpha-galactosidase

Abstract

Purified coffee bean alpha-galactosidase (alpha Gal) has been used for removing terminal alpha-galactose residues from the glyco-conjugates at the red cell surface, in studies of blood group conversion. Here, we report the isolation and sequence of the full-length clone for coffee bean alpha Gal by using the polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques. The cDNA clone (1.4 kb) contains a single open reading frame which encodes a protein of 378 amino acids (aa). Its authenticity is confirmed by perfect alignment of aa sequences obtained from purified coffee bean alpha Gal, and by immune reaction with the antibody raised against the enzyme. Furthermore, the protein produced in insect cells shows enzymatic activity towards a synthetic alpha Gal substrate, p-nitro-phenyl-alpha-galactopyranoside.