Study of the functional domains of β1-4Galactosyl transferase through the expression of the deletion constructs of cDNA in <italic>E. coli</italic> and mammalian cells: disulfide bond between CYS134 and CYS247 is required for folding and enzyme activity while the transmembrane domain is essential for stable expression in mammalian cells.

Abstract

β1,4Galactosyltransferase (β1,4GT) belongs to the family of glyeosyltransferases and transfers galactose from UDP-galactose to N-acetylglucosamine to produce N-acetyllactosamine with a β1-4 linkage[1]. Bovine β1,4GT is a 402 residue long protein, which has a general topology similar to other glycosyltransferases and type II transmembrane proteins that consists of a short amino-terminal cytoplasmic tail. a signal anchor domain, a stem region and a carboxyl-terminal catalytic domain. It has been localized in the trans-cisternae of the Golgi apparatus and has also been detected on the plasma membrane of several cell types. To delineate the function of various regions of bovine β1 AGT, we prepared cDNA constructs coding for the 402 residue long protein and its amino-terminal deleted forms in pGEX-2T vector, and expressed them in E. coli as glutathione-S-transferase (GST) fusion proteins. Recombinant proteins were localized in inclusion bodies which were solubilized in 5 M guanidine-HC 1. Renaturation and regeneration of the enzyme activity from the solubilized protein was strictly dependent on the presence of an “oxido-shuffling” reagent, a mixture of 8:1 mM reduced:oxidized glutathione. Renaturedireoxidized fusion proteins, purified on glutathione-affinity columns, were active in β1 AGT and lactose synthetase (LS) assays. After thrombin cleavage of the fusion protein and subsequent removal of the GST domain, the recombinant fj1 AGT has a specific activity of 50-70% as compared to that of bovine milk enzyme. The Km of the recombinant protein for N-acetylglucosamine and UDP-galactose is similar to that of bovine milk β1 AGT. Deletion analyses show that even after removal of the first 130 residues, both β1 AGT and LS activities remain intact.