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

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