Investigating Binding Characteristics
These investigations require the use of soluble ZFPs, since attachment to phage can alter the binding characteristics of the zinc fingers. Once a ZFP has been selected, its sequence is subcloned from pComb3H into a modified bacterial expression vector, pMal-c2, linking it to a sequence coding the maltose binding protein. The recombinant is then transformed into XL1-Blue cells and expression is induced by the addition of isopropyl β-D-thiogalactoside (IPTG). Freeze/thaw extracts may then be purified for use in the following experiments. Whilst purification is not necessary for multitarget ELISA, it is essential for measuring binding affinity by plasmon resonance and DNase footprints. It can be performed using a Heparin-Sepharose FPLC column equilibrated with zinc buffer followed by confirmation of homogeneity by SDS PAGE gel densitometry The same techniques are used to examine the binding properties of completed polydactyl ZFP chimera
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