Construction of an expression system for human alpha(1)-acid glycoprotein in E. coli: The roles of oligosaccharide moieties in structural and functional properties.

Unglycosylated recombinant human alpha(1)-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.