Construction of an Expression System for Human α₁-Acid Glycoprotein in E. coli: the Roles of Oligosaccharide Moieties in Structural and Functional Properties

Abstract

  Unglycosylated recombinant human α₁-acid glycoprotein (hAGP) variants (rF1^*S and rA) were prepared in an E. coli expression system using the Origami B strain and pET-3c vector. Thioredoxin was co-expressed to promote the appropriate folding of hAGP. SDS-PAGE under reducing conditions showed that rF1^*S and rA migrate as single bands after purification. However, several bands derived from rA were observed under non-reducing conditions because of the high reactivity of a free cystein residue (C149). We therefore prepared a mutant of A variant (C149R-A), and confirmed that this mutant maintained homogeneity. Circular dichroism and intrinsic tryptophan fluorescence spectroscopic analyses indicated that rF1^*S and C149R-A have almost the same conformational structures as F1^*S and A purified from serum. Ligand binding experiments using propranolol as a F1^*S ligand and disopyramide as an A specific ligand indicated that the capacity of rF1^*S and C149R-A is equivalent to those ligands as well as F1^*S and A from serum. These results suggest that the oligosaccharide moieties of hAGP have negligible effects on the structural and ligand binding properties of hAGP. Thus, rF1^*S and C149R-A promise to be useful in studies on the drug binding sites of hAGP.