SCREENING ASSAY FOR THE DETECTION OF THE PROTEIN-PROTEIN INTERACTION BETWEEN HIV-1 NEF PROTEIN AND THE SH3 DOMAIN OF HCK

The interaction between the Human Immunodeficiency Virus Nef protein ( HIV-1 Nef) and the Src Homology Region 3 (SH3) domain of Hck was studi ed using scintillation proximity assay (SPA), SPA is a quick and sensi tive method that does not require a separation step, thus allowing ass ays to be performed in a homogeneous environment, In contrast to most conventional techniques, SPA may also be used to detect low affinity p rotein-protein interactions, In this study, the assay was configured u sing biotinylated Hck SH3 domain expressed both as a GST fusion protei n and synthesized chemically in its' native form, Biotinylated Hck pro tein was immobilized to streptavidin-coated fluoromicrosphere SPA bead s and the binding of [H-3]Nef was detected by scintillation counting, Analysis of binding yielded an average equilibrium dissociation consta nt (K-D) of 183 +/- 30 nM for the interaction in line with reported va lues by other methods, The data presented demonstrates that using SPA, protein-protein interactions of relatively low affinity can be detect ed with a high degree of sensitivity and screening studies of inhibito rs of these associations could be facilitated by the high sample throu ghput achievable with SPA.