Development of annexin V mutants suitable for labeling with Tc(i)-carbonyl complex.

Abstract

(99m)Tc-annexin V can be used to image organs undergoing cell death during cancer chemotherapy and organ transplant rejection. We investigated whether the novel Tc-carbonyl labeling method would be suitable for annexin V. Two mutant molecules of annexin V, called annexin V-122 and annexin V-123, were constructed with N-terminal extensions containing either three or six histidine residues. These molecules were expressed cytoplasmically in E. coli and purified with a final yield of 33 mg of protein/L of culture. Analysis by SDS-PAGE, isoelectric focusing, gel filtration chromatography, and mass spectrometry confirmed the purity and homogeneity of the protein preparations. Both mutant proteins retained full binding affinity for cell membranes with exposed phosphatidylserine. Using the Tc-carbonyl reagent, both proteins could be labeled with (99m)Tc to specific activities of at least 10-20 microCi/microg with full retention of bioactivity. The radiolabeled proteins were stable when incubated with phosphate-buffered saline or serum in vitro, and there was no transchelation of label to serum proteins during in vitro incubation. In conclusion, annexin V can be modified near its N-terminus to incorporate sequences that form specific chelation sites for (99m)Tc-carbonyl without altering its high affinity for cell membranes.