A SINGLE FC BINDING DOMAIN-ALKALINE PHOSPHATASE GENE FUSION EXPRESSESA PROTEIN WITH BOTH IGG BINDING ABILITY AND ALKALINE-PHOSPHATASE ENZYMATIC-ACTIVITY

A recombinant gene fusion was created and cloned using a previously co nstructed gene encoding a monodomain IgG Fc binding protein and the ge ne coding for bacterial alkaline phosphatase. The construct was able t o express and secrete a fusion protein that exhibited both IgG binding and alkaline phosphatase enzymatic activities. Greater than 60% of th e protein demonstrating both biological activities was detected from p eriplasmic space preparations. Nanogram concentrations of the Fc bindi ng-alkaline phosphatase fusion protein allowed primary IgG antibody de tection without the use of conjugated secondary antibodies. Removal of the domain coding for alkaline phosphatase resulted in decreased resi stance of the protein to proteolytic degradation and the loss of IgG F c binding ability. Using affinity-purified fusion protein, the specifi city of binding to IgG, IgM and IgA was examined; binding was strong t o IgG and barely detectable against IgM or IgA. Affinity for binding o f the fusion protein to IgG (K-d = 6.7X10(-8) M) was determined to be equal to or greater than previously reported for protein A.