Expression of deletion constructs of bovine β-1, 4-galactosyltransferase in Escherichia coli: importance of Cysl34 for its activity

Abstract

Bovine β-1, 4-galactosyltransferase (β-1, 4-GT; EC 2.4.1.90) belongs to the glycosyltransferase family and as such shares a general topology: an N-terminal cytoplasmic tail, a signal anchor followed by a stem region and a catalytic domain at the C-tenninal end of the protein. cDNA constructs of the N-terminal deleted forms of β-1, 4-GT were prepared in pGEX-2T vector and expressed in E.coli as glutathione-S-transferase (GST) fusion proteins. Recombinant proteins accumulated within inclusion bodies as insoluble aggregates that were solubilized in 5 M guanidine HCl and required an ‘oxido-shuffling’ reagent for regeneration of the enzyme activity. The recombinant (β-1, 4-GT, devoid of the GST domain, has 30–85% of the sp. act. of bovine milk β-1, 4-GT with apparent K_ms for N-acetylglucosamine and UDP-galactose similar to those of milk enzyme. Deletion analysesshow that both (β-1, 4-GT and lactose synthetase activities remain intact even in the absence of the first 129 residues (pGT-dl29). The activities are lost when either deletions extend up to residue 142 (pGT-dl42) or Cysl34 is mutatedto Ser (pGT-dl29C134S). These results suggest that the formation of a disulfide bond involving Cysl34 holds the protein in a conformation that is required for enzymaticactivity.