Genetic and chemical modification of proteins for site-specific immobilization and binding assays.

摘要

Two strategies have been employed to modify proteins by recombinant DNA technology to facilitate their site-specific immobilization. One approach is introducing a specific amino acid residue (e.g., cysteine) with a reactive side-chain functional group at a pre-defined location of the protein by site-directed mutagenesis. The feasibility and advantages of this approach were demonstrated using the proteinase subtilisin BPN;A homogeneous bioluminescence binding assay for the vitamin folate was developed based on a chemically conjugated glucose-6-phosphate dehydrogenase (G6PDH) and the bacterial luciferase system. By coupling the enzymatic reactions of G6PDH and the bacterial luciferase system, the activity of G6PDH was measured through the bioluminescence generated in the coupled reactions. This led to the improvement of the detection limit of G6PDH, which in turn resulted in a better detection limit of the homogeneous assay for folate using G6PDH as the label.;The genetically modified photoprotein aequorin was used in the development of a binding assay for biotin. A biotinylated aequorin conjugate was prepared using an in vivo posttranslational modification process. The aequorin prepared has one biotin molecule attached at a specific site of the protein. A heterogeneous binding assay for biotin was developed using this biotinylated aequorin and streptavidin-coated magnetic particles.