Expression, refolding, and isotopic labeling of human serum albumin domains for NMR spectroscopy.
Many different compounds bind to human serum albumin (HSA), which can be a significant problem in the drug discovery process. To aid in the design of drug molecules that do not bind to HSA, the structures of HSA/ligand complexes would be very useful. However, little information has been reported on the structures of small molecules complexed to HSA. In this paper, we describe a procedure for preparing isotopically labeled domains of HSA for nuclear magnetic resonance (NMR) studies. The procedure involves the expression in Escherichia coli, refolding, and a multistep purification. Domains I and III are capable of folding into stable structural units and producing well resolved (15)N/(1)H correlation spectra, whereas domain II forms significant aggregates at sub-millimolar concentration. Using our protocols, isotopically labeled and properly folded domains I and III can be effectively produced in large quantities for NMR-based structural studies and NMR-based screening. This provides a valuable tool for obtaining structural information on HSA/ligand complexes by NMR which will be useful in drug discovery.
Mao
H
H
Pharmaceutical Discovery Division, Abbott Laboratories, Abbott Park, Illinois 60044, USA.
Gunasekera
A H
AH
Fesik
S W
SW
D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012709:Serum Albumin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D012709:Serum Albumin