SPECIFIC DETECTION OF HIS-TAGGED PROTEINS WITH RECOMBINANT ANTI-HIS TAG SCFV-PHOSPHATASE OR SCFV-PHAGE FUSIONS

Using a cell-bound immunogen, we have generated a monoclonal antibody, 3D5, that recognizes carboxy-terminal oligo-histidine tags (His tags) on a wide variety of proteins. From this monoclonal antibody, we have generated a single-chain fragment of the variable domains (scFv), a d imeric scFv-alkaline phosphatase fusion and an oligovalent scFv-displa y phage. The antibody in its various formats is an effective tool used in fluorescence-activated cell sorting analysis, the BIAcore(R) metho d, Western blots and enzyme-linked immunosorbent assay (ELISA). Wester n blots and ELISAs can be developed directly by using crude extracts o f E. coli cells that produce the scFv-alkaline phosphatase fusion, thu s providing an inexhaustable and convenient supply of detection reagen t. Alternatively, oligovalent scFv-displaying phage can be used direct ly from culture supernatants for this purpose. The dissociation consta nts, K-D, of the peptide KGGHHHHH (K-D = 4 x 10(-7) M) and of imidazol e (K-D = 4 x 10(-4) M) were determined. Molecular modeling of the Fv f ragment suggests the occurrence of two salt bridges between the proton ated histidine side chains of the peptide and the acidic groups in the antibody, explaining why the antibody or the substrate may be eluted under mildly basic conditions.