Expression of high-affinity human antibody fragments in bacteria.

Romain Rouet, David Lowe, Kip Dudgeon, Brendan Roome, Peter Schofield, David Langley, John Andrews, Peter Whitfeld, Lutz Jermutus, Daniel Christ

Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales, Australia.

Nature protocols 2012 Feb

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Here we describe protocols for the expression of human antibody fragments in Escherichia coli. Antigen-specific clones are identified by soluble fragment ELISA and concentrated by periplasmic preparation. They are then further purified by affinity chromatography. This article provides an overview of expression and purification strategies for human antibody fragments, as well as detailed protocols for the identification of high-affinity binders and for affinity maturation.

Citation

Romain Rouet, David Lowe, Kip Dudgeon, Brendan Roome, Peter Schofield, David Langley, John Andrews, Peter Whitfeld, Lutz Jermutus, Daniel Christ. Expression of high-affinity human antibody fragments in bacteria. Nature protocols. 2012 Feb;7(2):364-73