Affinity cleavage and targeted catalysis of proteins using the avidin-biotin system.
The avidin-biotin system was used in order to target enzymes to their substrates in complex mixtures of proteins in solution. The approach described here thus mimics natural systems in which enzymes usually act in selective fashion, due, perhaps, to proximity effects. For affinity cleavage studies, biotinyl transferrin was used as a model target substrate. Avidin or streptavidin was then employed to bridge between the biotinylated target protein and a biotinyl protease. Bovine serum albumin was included in the reaction mixtures to assess the level of nonspecific cleavage. In the case of an unbiotinylated target protein, avidin could be used to inhibit the hydrolytic action of the biotinyl protease. In some systems, a biotinyl antibody could be used to direct the avidin-bridged biotinyl protease to an unbiotinylated target antigen. The data support the contention that preferential cleavage reflects two separate phenomena: (i) avidin confers a conformational alteration of the biotinylated target protein, and (ii) the biotinyl protease is targeted (via the avidin bridge) to the proximity of the biotinylated target protein, thereby promoting cleavage of the conformationally altered molecule. This is the first report in which a proteolytic enzyme could be selectively targeted to specifically hydrolyze a defined protein substrate in solutions containing a complex mixture of other proteins. The approach appears to be a general phenomenon for "targeted catalysis", appropriate for other applications, particularly for affinity cleavage and targeted catalysis of cell-based macromolecules.
Bayer
E A
EA
Department of Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Grootjans
J J
JJ
Alon
R
R
Wilchek
M
M
D000345:Affinity Labels; D001360:Avidin / Q000494:pharmacology*; D001710:Biotin; D002384:Catalysis; D002918:Chymotrypsin / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D007700:Kinetics; D008961:Models, Structural; D011506:Proteins / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D011506:Proteins; D014168:Transferrin; D001360:Avidin; D012710:Serum Albumin, Bovine; D001710:Biotin; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin