Purification and Characterization of His-Tagged Antibody Fragments

Immobilized metal affinity chromatography (IMAC) has become a widely used method for the affinity purification of recombinant proteins and, in particular, of antibody fragments produced in Escherichia coli . IDA-Sepharose and NTA-agarose charged with different divalent cations such as Ni(II), Cu(II), or Zn(II) are the chelating media most commonly used. The metal ions are bound by these chelator groups, leaving vacant coordination sites for the interaction with an oligo-histidine peptide, typically His₆ , fused to the recombinant protein. In the case of antibody fragments that are composed of different chains, e.g., Fv and Fab, the site of attachment for the His₆ tag has to be carefully chosen, and mild chromatographic conditions must be applied in order to recover the functional hetero-dimeric protein. Here, we describe a protocol that enables the production and purification of His-tagged antibody fragments from bacterial cell lysates using IMAC, as well as their analysis in ELISA and on Western blots using Ni/NTA-alkaline phosphatase conjugate as a secondary reagent. Thus, the His₆ -tag, offers a versatile tool for the rapid isolation of bacterially produced antibody fragments, enabling their biochemical analysis and/or application in various assays.