Expression of deletion constructs of bovine beta-1,4-galactosyltransferase in Escherichia coli: importance of Cys134 for its activity
Boeggeman, E.E.; Balaji, P.V.; Sethi, N.; Masibay, A.S.; Qasba, P.K.
Protein Engineering 6(7): 779-785
1993
ISSN/ISBN: 0269-2139 PMID: 8248102 Document Number: 417579
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-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 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 K-ms 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 protein in a conformation that is required for enzymatic activity.