PEDS Advance Access published online on October 24, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm034
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Folding of an antibody variable domain in two functional conformations in vitro: calorimetric and spectroscopic study of the anti-ferritin antibody VL domain
1 Institute of Bio-Organic Chemistry, National Academy of Sciences of Belarus, Minsk 220141, Belarus 2 Research and Production Center for Hematology and Transfusiology, Ministry of Health of the Republic of Belarus, Minsk 220053, Belarus
4 To whom correspondence should be addressed. E-mail: tsybovs{at}musc.edu
Understanding refolding pathways of recombinant antibody fragments is essential for efficient production of these proteins of high biomedical significance. The recombinant VL domain of mouse anti-human ferritin antibody F11 formed two distinct functional conformations obtained by refolding from bacterial inclusion bodies using two different procedures. Involvement of a dialysis step at pH 2–3 resulted in the VL-1 conformation with fluorescence of the highly conserved Trp-35 residue quenched by the spatially proximal disulfide bond. This conformation was identical to the ‘native’ VL domain folded in host cells and purified from the cytoplasm. In the absence of the acidic dialysis step, the VL domain adopted a previously unreported conformation, VL-2, that demonstrated prominent fluorescence due to a local structural disorder around Trp-35. Furthermore, VL-2 showed changes in secondary structure and significantly lower stability as determined by differential scanning calorimetry and denaturant-induced unfolding. While more flexible VL-2 binds human ferritin both in solution and after surface adsorption of the antibody domain, the VL-1 conformer needs an adsorption-induced conformational change to allow the access of ferritin to the antigen-binding site. Noteworthy, the two macroscopic conformations constitute kinetically trapped dimers and do not interconvert at elevated temperatures (3 weeks at 37°C or 15 min at 60°C), which indicates a high energetic barrier between them. As a major finding, this paper provides the first description for two stable and functional conformations of an antibody domain.
Keywords: conformational stability/conformers of folding/protein refolding/recombinant antibody/VL domain
Received February 15, 2007; revised June 4, 2007; accepted June 5, 2007.
3 Present address: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.