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Screening Assay for the Detection of the Protein-Protein Interaction Between HIV-1 Nef Protein and the SH3 Domain of Hck

Nycomed Amersham plc, Cardiff Laboratories, Forest Farm Estate, Whitchurch, CF4 7YT, UK

Kalle Saksela

Institute of Medical Technology, University of Tampere, P.O. Box 607, Fin-33101, Tampere, Finland

Stephen M. Game

Nycomed Amersham plc, Cardiff Laboratories, Forest Farm Estate, Whitchurch, CF4 7YT, UK

Gerard O'Beirne

Nycomed Amersham plc, Cardiff Laboratories, Forest Farm Estate, Whitchurch, CF4 7YT, UK

Neil D. Cook

Nycomed Amersham plc, Cardiff Laboratories, Forest Farm Estate, Whitchurch, CF4 7YT, UK

The interaction between the Human Immunodeficiency Virus Nef protein (HIV-1 Nef) and the Src Homology Region 3 (SH3) domain of Hck was studied using scintillation proximity assay (SPA). SPA is a quick and sensitive method that does not require a separation step, thus allowing assays to be performed in a homogeneous environment. In contrast to most conventional techniques, SPA may also be used to detect low affinity protein-protein interactions. In this study, the assay was configured using biotinylated Hck SH3 domain expressed both as a GST fusion protein and synthesized chemically in its' native form. Biotinylated Hck protein was immobilized to streptavidin-coated fluoromicrosphere SPA beads and the binding of [³H]Nef was detected by scintillation counting. Analysis of binding yielded an average equilibrium dissociation constant (KD) of 183 ± 30 nM for the interaction in line with reported values by other methods. The data presented demonstrates that using SPA, protein-protein interactions of relatively low affinity can be detected with a high degree of sensitivity and screening studies of inhibitors of these associations could be facilitated by the high sample throughput achievable with SPA.

Journal of Biomolecular Screening, Vol. 3, No. 1, 37-41 (1998)
DOI: 10.1177/108705719800300105


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