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A model system to study the structural stability of engineered antibodies: mutants of a SCFV specific to AMCV

Author:
Galeffi, P., d'Apolito, M., Lombardi, A., Cavicchioni, G., Manicino, T., Sperandei, M., Cantale, C.
Source:
Acta horticulturae 2004 no.660 pp. 615-620
ISSN:
0567-7572
Subject:
Artichoke mottled crinkle virus, Escherichia coli, antibodies, bacteria, cytoplasm, databases, enzyme-linked immunosorbent assay, genes, germ cells, molecular models, mutants, viruses
Abstract:
It had been proved previously that the single chain variable fragment of the anti-viral antibody scFv alphaAMCV (Artichoke Mottled Crinkle Virus) (F8) is functionally expressed even in the reducing environment of both bacteria and plant cytoplasm and is endowed intrinsically with high thermodynamic stability. In order to investigate the structural determinants of this scFv, we analysed its primary sequence and compared it with the databanks available on antibodies and variable fragments, both of the crystallised and sequenced type. The result was that the germline genes encoding F8 most closely match the kappa chain V(K)21.C gene and the heavy chain V(H)68.5N from V(H)7183 gene family (99% identity). Two amino-acid residues of the framework region, borderline to the second hypervariable loop (CDR2) for V(H), appeared particularly interesting and were mutagenised. In total three mutants, including the double mutant, were designed, constructed and produced in Escherichia coli. Their functionality was tested by ELISA and immuno-printing, using plant materials infected by AMCV. One of the mutants shows an improved ability in detecting the virus in comparison with wild type scFv, the second one completely loses this ability, while the double mutant retain a very low affinity. Molecular models of the three mutants and wild type scFvs were built by a combination of protein homology modelling and ab initio modelling. With the study and the comparison of all these models it was possible to explain the behaviour observed and this will be the basis for further modifications aimed at a better understanding of F8’s particular features.
Agid:
318932