Protease Sensing Cut-N-Glo Mapping & Diagnostic Tool
Cut-N-Glow is the first fully, biological in vivo protease mapping tool that emits fluorescence. Our assay is easily tailored via standard cloning techniques to detect different proteases or to map protease specificity. No chemical synthesis is required therefore there is no need for co-factors or co-substrates. Additionally, this assay only requires two reagents and both are proteins that can be easily obtained following over-expression of E. coli. In addition to the advantage of emitting a fluorescent signal in the presence of proteases, Cut-N-Glow, as its name implies, is designed with a conditional distortion to convert self-assembling GFP proteins into a site specific protease switch. Our approach to the distortion is reversible through proteolysis by constraining the N and C termini of GFP 11 with a protease-sensitive tether, eglin C. The feature distinguishing our system from other GFP reporters is that there is a gain of fluorescence, rather than a loss of fluorescence in proteolysis both in vitro and in vivo.
Proteases occur naturally in all organisms and are valuable tools in medical diagnostics serving as initiators of cell signaling, as regulators of immune responses, and as agents of infectious disease. Therefore, mapping proteases in parasitic diseases and bacteria as well as assayable proteases associated with cancer could lead to the identification of shared structural similarities validating potential drug targets. Our strategy utilized split proteins in a conditionally inactive form with the aid of a conformational distortion maintained by a cleavable tether. We applied this method to convert split GFP into a latent fluorophore that can be activated by site-specific proteolysis. The chimeric GFP served as substrate for representative enzymes from the three major protease classes: serine, cysteine, and aspartic acid.
Human clinical diagnostic protease detection for diseases & infection:
- In bacterial infections such as M.tuberculosis, C. botulism and MARTX toxins such as V. Cholera.
- In assayable proteases associated with cancer such as human kallikrein-3, commonly known as prostate specific antigen (PSA).
- Targeting the HIV protease, for the AIDS virus.
- In matrix metalloproteinases’s (MMPs) involved in tissue remodeling such as morphogenesis, angiogenesis, cirrhosis and arthritis.
- In parasitic infections such as Cryptosporidium parvum, Plasmodium falciparum, Schistosomiasis, and Trypanosoma cruzi.
Potential utility as a diagnostic and as a research tool in vitro or in vivo:
- Identification of peptide sequences cleaved by a particular protease (substrate discovery)
- Identification of the protease responsible for cleaving a specific peptide sequence (protease discovery)
- Identification of protease variants, created through mutation, that cleave at a user-defined peptide sequence (protease evolution)
- Detection of a characterized protease in chromatographic fractions or laboratory buffers (protease detection).
- Great potential to be implemented as an in vivo technique, to stand alone as the first fully biological, gain-of-fluorescence protease mapping tool.
- Features a highly stable output signal. Our results indicate that the fluorescent signal that follows site-specific proteolysis S/N level – despite the presence of E. coli lysate.
- Efficient and affordable. This assay only requires two reagents and both are proteins that can be easily obtained following over-expression of E. coli.
- No chemical synthesis is required.
- No need for co-factors or co-substrates.
- Easily tailored assay via standard cloning techniques to detect different proteases or to map protease specificity.
- Effectively adapted to portable field testing.
State of Development
Diagnostic assay available by distribution from Sandia Biotech.
Provisional patent 61/514,074
Brian Callahan, Ph.D.
Brian Callahan obtained his B.Sc. from the State University of New York in Cortland (1996). He worked as a laboratory technician (1997-1999), before pursuing a Ph.D. in Biochemistry and Biophysics at the University of North Carolina in Chapel Hill (2000-2005). In 2006, Brian received an NIH-funded fellowship in Biodefense and Emerging Infectious Diseases from the Wadsworth Center in Albany, NY, where he is currently a postdoctoral associate in Professor Marlene Belfort’s laboratory.
Marlene Belfort, Ph.D.
Marlene Belfort is a distinguished scientist and has won many awards and honors within the scientific community. She directs her own laboratory at the New York State Department of Health, Wadsworth Center, called The Belfort Laboratory. She is an Adjunct Scientist for the Marine Biological Laboratory in Woods Hole, MA and founding member of Mobile DNA Cluster; an Adjunct Professor in the Department of Chemical and Biological Engineering & the Department of Biology with Rensselaer Polytechnic Institute in Troy, NY; an Adjunct Professor for the Biology Department with SUNY at Albany in Albany, NY; a Professor for the School of Public Health at SUNY at Albany and the New York State Department of Health. Marlene Belfort holds two patents for intein technology and recently applied for a third.
Awards & Honors
- Distinguished Scientist, Wadsworth Center, Molecular Genetics
- Fellow, American Association for Advancement of Science
- Doctor Philosophiae Honoris Causa, Hebrew University
- Distinguished Professor, School of Public Health, Biomedical Sciences
- Alice Evans Award, American Society for Microbiology
- MERIT Award NIH
- Fellow, American Academy of Microbiology
- Member, National Academy of Sciences (NAS)
- Fellow, American Academy of Arts and Sciences
- Ph.D., University of California at Irvine (1972)
- Postdoctoral training, Hebrew University, Jerusalem
Callahan, Brian P., Stanger, Matthew, J., Belfort, Marlene: Protease Activation of Split Green Fluorescent Protein: ChemBioChem – Chemistry Clinical Biochemistry 10-13-2010DOI: 10.1002/cbic.201000453
Diane L. Borghoff, B.S., M.S.
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