Fluorescence anisotropy assay for proteolysis of specifically labeled fusion proteins.
A cloning method and plasmid vectors that permit fluorescence-anisotropy-based measurement of proteolysis are reported. The recombinant protein substrates produced by this method contain a tetracysteine motif that can be site-specifically labeled with bis-arsenical fluorophore [Science 281 (1998) 269]. Six protein substrates with an N-terminal fusion of the tetracysteine motif and different protease recognition sites were created and tested for reaction with commercial proteases commonly used to process recombinant fusion proteins. In each case, proteolysis of a single susceptible peptide bond could be monitored in real time and with sufficient data quality to allow numerical analysis of proteolysis reaction kinetics. Measurement of proteolysis extent using fluorescence anisotropy is shown to be comparable to densitometry measurements made on denaturing polyacrylamide gels but with the added advantages implicit in a time-resolved measurement, quantification by a spectroscopic measurement, and facile extensibility to high-throughput formats. The assay was also demonstrated as a general tool for monitoring proteolysis of multidomain fusion proteins containing an internal protease site such as are being created in structural genomics studies worldwide.
Blommel
Paul G
PG
The University of Wisconsin Center for Eukaryotic Structural Genomics, Biophysics Graduate Degree Program and Biochemistry Department, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706, USA.
Fox
Brian G
BG
D020816:Amino Acid Motifs; D001483:Base Sequence; D002352:Carrier Proteins / Q000032:analysis*; D010450:Endopeptidases; D004765:Enteropeptidase / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D005454:Fluorescence Polarization; D058619:Maltose-Binding Proteins; D010447:Peptide Hydrolases / Q000032:analysis*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000032:analysis*; D013917:Thrombin / Q000378:metabolism
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; D010447:Peptide Hydrolases; C099488:TEV protease; D013917:Thrombin; D015951:Factor Xa; D004765:Enteropeptidase