Health Research, Inc. Announces Cut-N-Glow as a New Protease Sensing, Mapping and Detection Tool
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Cut-N-Glow triggers a green fluorescent “glow” when pathogens are present. Light emission can be followed quantitatively using a fluorimeter, or qualitatively by eye as the assay solution turns green.
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.
"Our assay is easily tailored via standard cloning techniques to detect proteases of varying specificity, to identify protease inhibitors, or to map protease substrate preference in vitro or in vivo," said Brain Callahan Ph.D. "It's also efficient and affordable. No chemical synthesis co-factors or co-substrates are required. You need only two reagents, and both are proteins that can be easily obtained following over-expression of E. coli."
With its operational simplicity and in vitro/in vivo versatility, the Cut-N-Glow system has great potential to be implemented as a human clinical diagnostic for protease based disease detection and as a tool for protease research.
Human clinical diagnostic applications include: 1) Detecting protease-based disease in microbial infections, such as M. tuberculosis, Clostridium botulinum, Vibrio Cholera, Cryptosporidium parvum, Plasmodium falciparum, and Trypanosoma cruzi; 2) Detecting secreted proteases associated with cancer such as human kallikrien-3, commonly known as prostate specific antigen (PSA); 3) Sensing the HIV protease, for monitoring AIDS therapy; and 4) Detecting cell-associated matrix metalloproteinases's (MMPs) involved in tissue remodeling such as morphogenesis, angiogenesis, cirrhosis and arthritis.
Potential utility as a tool for protease research include: 1) Identification of peptide sequences cleaved by a particular protease (substrate discovery); 2) Identification of the protease responsible for cleaving a specific peptide sequence (protease discovery); 3) Identification of protease variants, created through mutation, that cleave at a user-defined peptide sequence (protease evolution); and 4) Detection of a characterized protease in chromatographic fractions or laboratory buffers (protease detection).
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.
"Our assay is easily tailored via standard cloning techniques to detect proteases of varying specificity, to identify protease inhibitors, or to map protease substrate preference in vitro or in vivo," said Brain Callahan Ph.D. "It's also efficient and affordable. No chemical synthesis co-factors or co-substrates are required. You need only two reagents, and both are proteins that can be easily obtained following over-expression of E. coli."
With its operational simplicity and in vitro/in vivo versatility, the Cut-N-Glow system has great potential to be implemented as a human clinical diagnostic for protease based disease detection and as a tool for protease research.
Human clinical diagnostic applications include: 1) Detecting protease-based disease in microbial infections, such as M. tuberculosis, Clostridium botulinum, Vibrio Cholera, Cryptosporidium parvum, Plasmodium falciparum, and Trypanosoma cruzi; 2) Detecting secreted proteases associated with cancer such as human kallikrien-3, commonly known as prostate specific antigen (PSA); 3) Sensing the HIV protease, for monitoring AIDS therapy; and 4) Detecting cell-associated matrix metalloproteinases's (MMPs) involved in tissue remodeling such as morphogenesis, angiogenesis, cirrhosis and arthritis.
Potential utility as a tool for protease research include: 1) Identification of peptide sequences cleaved by a particular protease (substrate discovery); 2) Identification of the protease responsible for cleaving a specific peptide sequence (protease discovery); 3) Identification of protease variants, created through mutation, that cleave at a user-defined peptide sequence (protease evolution); and 4) Detection of a characterized protease in chromatographic fractions or laboratory buffers (protease detection).
