Proteases that can distinguish among different post-translational forms of tyrosine engineered using multicolor flow cytometry.
We report the isolation of a highly active (k(cat)/K(M) approximately 10(5) M(-1)s(-1)) variant of the E. coli endopeptidase OmpT that selectively hydrolyzes peptides after 3-nitrotyrosine while effectively discriminating against similar peptides containing unmodified tyrosine (160-fold), sulfotyrosine (3600-fold), phosphotyrosine (>8000-fold), and phosphoserine (>8000-fold). The isolation of endopeptidase variants that can discriminate between substrates based on the post-translational modification of Tyr was made possible by implementing a multicolor flow cytometric assay for the screening of large mutant libraries. For the multicolor assay, a desired selection substrate was used simultaneously with multiple counterselection fluorescent substrates to isolate rare enzyme variants that displayed finely tuned substrate specificity. This work demonstrates that enzymes with exquisite selectivity can be isolated from large libraries using appropriate high throughput screening approaches and constitutes a critical step toward the production of a 3-nitrotyrosine-specific protease useful for proteomic applications.
Varadarajan
Navin
N
Department of Chemical Engineering, University of Texas at Austin, Austin, Texas 78712, USA.
Pogson
Mark
M
Georgiou
George
G
Iverson
Brent L
BL
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D005434:Flow Cytometry; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D019151:Peptide Library; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D019151:Peptide Library; D010455:Peptides; C499588:ompT protein, E coli; C002744:3-nitrotyrosine; D014443:Tyrosine; D010447:Peptide Hydrolases