In vitro evolution of the binding specificity of neocarzinostatin, an enediyne-binding chromoprotein

Neocarzinostatin is the most studied member of the enediyne-chromoprotein family, and is clinically used as an antitumoral agent. Neocarzinostatin could be a promising drug delivery vehicle if new binding specificities could be conferred to its protein scaffold. We used in vitro evolution methods to demonstrate that this approach is feasible. We created large libraries containing between 1.7 x 10(8) and 1.4 x 10(9) independent clones, where up to 13 side chains pointing toward the binding crevice were randomly substituted. We then used phage display to select variants that bind to a model ligand (testosterone) which is unrelated to the natural ligand of neocarzinostatin. Several different binders were selected from each library. The corresponding proteins were expressed in Escherichia coli and their affinities and specificities were characterized in detail. K(D) values of about 20 nM were obtained for streptavidin-bound testosterone. The K(D) of selected proteins for free soluble testosterone are between 7 and 55 microM and therefore higher than the K(D) for streptavidin-bound testosterone. The spacer and streptavidin used during selection contributed to the high affinity of the selected binders for the target. Binding studies of 15 different steroids related to testosterone allowed us to determine that C3, 4, 5, 6, and 7 on cycles A and B and the conjugated 3 oxo group of the steroid molecule were essential for molecular recognition. Other testosterone analogues substituted on C1, 2, 9, 11, 15, and 17 were not discriminated from testosterone. These results demonstrate that the binding specificity of this protein family can be extended to compounds that are completely unrelated to the natural enediyne chromophore family. This type of highly expressed, stable proteins with tailored binding properties have a wide potential range of applications.