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Expression of high-affinity human antibody fragments in bacteria.

Nat Protoc.. 2012-02;  7(2):364-73
Rouet R, Lowe D, Dudgeon K, Roome B, Schofield P, Langley D, Andrews J, Whitfeld P, Jermutus L, Christ D. Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales, Australia.
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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.