Single-step purification of a bacterially expressed antibody Fv fragment by immobilized metal affinity chromatography in the presence of betaine.

A procedure was developed for the rapid isolation of an antibody Fv fragment expressed in Escherichia coli via immobilized metal affinity chromatography. Metal affinity was mediated by fusing hexahistidine tails to both the VL and the VH domain and was thus independent of the antigen-binding specificity. Unexpectedly, it was not possible to isolate the Fv fragment with correct stoichiometric composition of the two variable domains under standard chromatographic conditions. Proper non-covalent association of VL and VH was, however, maintained when using glycine betaine as electrolyte, thus permitting purification of the intact Fv fragment to homogeneity in a single step.