Elizabeth E. Boeggeman, Petety V. Balaji, Navin Sethi, Arni S. Masibay and Pradman K. Qasba. Expression of deletion constructs of bovine beta-1, 4-galactosyltransferase in Escherichia coli: Importance of Cyse134 for its activity. Protein Engineering vol.6 no.7 pp. 779-785, 1993.
Bovine beta-1, 4-galactosyltransferase (beta-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-terminal end of the protein. cDNA constructs of the N-terminal deleted forms of beta-1, 4-GT were prepared in pGEX-2T vector and expressed in E.coli as glutathione-S-trnasferase (GST) fusion proteins. Recombinant proteins accumulated within inclusion bodies as insoluble aggregates that were solubilized in 5 M guanidine HCI and required an 'oxido-shuffling' reagent for regeneration of the enzyme activity. The recombinant beta-1, 4-GT devoid of the GST domain, has 30-85% of the sp.act. of bovine milk beta-1, 4-GT with apparent KmS for N-acetylglucosamine and UDP-galactose similar to those of milk enzyme. Deletion analyses show that both beta-1, 4-GT and lactose synthetase activities remain intact even in the absence of the first 129 residues (pGT-d129). The activities are lost when either deletions extend up to residue 142 (pGT-d142) or Cys134 is mutated to Ser (pGT-d129C134S). These results suggest that the formation of a disulfide bond involving Cys134 holds the formation of a disulfide bond involving Cys134 holds the protein in a conformation that is required for enzymatic activity.
