Refolding of scFv mini-antibodies using size-exclusion chromatography via arginine solution layer

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2009-07, Vol.877 (22), p.2045-2051
• Main Authors: Fursova, K.K., Laman, A.G., Melnik, B.S., Semisotnov, G.V., Kopylov, P.Kh, Kiseleva, N.V., Nesmeyanov, V.A., Brovko, F.A.
• Format: Article
• Language: English
• Subjects: Arginine; Arginine - chemistry; Chromatography, Gel - instrumentation; Chromatography, Gel - methods; Escherichia coli - genetics; Escherichia coli - metabolism; Immunoglobulin Variable Region - chemistry; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - isolation & purification; Immunoglobulin Variable Region - metabolism; Protein Folding; Refolding; Renaturation; scFv; Single-Chain Antibodies; Size-exclusion chromatography; Solutions - chemistry
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2009.05.038

The method for refolding of mini-antibodies using size-exclusion chromatography via arginine solution layer was developed. This method allows to refold scFv, to separate both aggregated protein and low molecular weight compounds and to isolate functionally active protein preparation in monomeric form. The comparison of various scFv preparations isolated either from inclusion bodies or from soluble fraction revealed that refolded mini-antibodies demonstrate higher antigen-binding activity. Mini-antibodies refolded in the presence of arginine also demonstrate higher electrophoretic mobility during native PAGE in comparison with soluble cytoplasmic antibodies. Both soluble as well as refolded antibodies had similar CD spectra. Refolded mini-antibodies are storage-stable.