Mapping protein-protein interactions with alkaline phosphatase fusion proteins

Abstract

This chapter describes a convenient and versatile method for determining the molecular recognition properties of SH3 domains, using bacterial alkaline phosphatase as a fusion protein. A variety of proteins or peptides have been fused to the N terminus of alkaline phosphatase (AP) by genetic engineering, including antibody chains and the extracellular domain of the steel receptor. With the ability to display a wide range of polypeptides at the N terminus of bacterial AP, it is possible to identify or map protein-protein interactions in a number of formats. There are several kinds of AP substrates available so a particular substrate can be chosen to suit specific needs. The utility of this system is enhanced by the fact the AP fusion protein is secreted from E. coli and the culture medium can be used directly in experiments once the appropriate fusions have been constructed, the cells are simply grown overnight, and the fusion protein is harvested with the culture supernatant. The successful application of the AP fusion system is to examine binding properties of the SH3 domain and other protein-binding modules. This system may prove useful for the general study of protein-protein interactions.