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.
Nishi
Koji
K
Department of Biopharmaceutics, Kumamoto University, Japan.
Fukunaga
Naoko
N
Ono
Tomomi
T
Akuta
Teruo
T
Yumita
Nagahiko
N
Watanabe
Hiroshi
H
Kadowaki
Daisuke
D
Suenaga
Ayaka
A
Maruyama
Toru
T
Otagiri
Masaki
M
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D005818:Genetic Engineering; D006801:Humans; D008024:Ligands; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D009961:Orosomucoid / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013329:Structure-Activity Relationship
D008024:Ligands; D009844:Oligosaccharides; D009961:Orosomucoid