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

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 for...

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Bibliographic Details
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
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Summary: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.
ISSN:1570-0232
1873-376X
DOI:10.1016/j.jchromb.2009.05.038