Methods, compositions, and kits for detection of bacterial proteases
AssigneeCity of Hope
InventorsMarkus Kalkum, Ismail Al-Abdullah
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Abstract
Provided herein are novel fluorogenic and bioluminescent bacterial protease substrates that contain at least one bacterial protease cleavage site. In some embodiments, the proteases that cleave the protease substrates are bacterial proteases including those used for tissue dissociation, cell isolation, and cell detachment, such as the preparation of islet cells. Additionally, the novel protease substrates described herein may be used to assess the activity of certain bacterial proteases involved in isolating primary cells and stem cells. Methods and protease detection kits using the novel protease substrates are also provided herein.
Claims38
A fluorogenic protease substrate comprising:
- (a) a donor fluorophore;
- (b) an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore;
- and (c) one or more amino acid sequences comprising at least one bacterial protease cleavage site, the amino acid sequence comprising:
- Ala-Gly-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Pro-Gly-Gly
- (i.e., SEQ ID NO:
- 1).
A method for detecting the presence of one or more bacterial proteases in a sample comprising:
- exposing the sample putatively containing the one or more bacterial proteases to an enrichment matrix comprising a fluorogenic protease substrate that is capable of eliciting a detectable fluorescence signal when modified by the one or more bacterial proteases, the fluorogenic protease substrate comprising one or more amino acid sequences comprising at least one bacterial protease cleavage site, the amino acid sequence comprising:
- Ala-Gly-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Pro-Gly-Gly
- (i.e., SEQ ID NO:
- 1);
- measuring the level of change in the detectable fluorescence signal;
- and
- detecting the presence of the one or more bacterial proteases when the level of change in the detectable fluorescence signal in the sample is elevated.
A bacterial protease detection kit comprising:
- an enrichment matrix comprised of one or more fluorogenic protease substrates comprising:
- (a) a donor fluorophore;
- (b) an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore;
- and (c) one or more amino acid sequences comprising at least one bacterial protease cleavage site, the amino acid sequence comprising:
- Ala-Gly-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Pro-Gly-Gly
- (i.e., SEQ ID NO:
- 1),
- wherein a detectable fluorescence signal is produced upon interaction of the substrate with the bacterial protease.
A bioluminescent protease substrate comprising one or more luciferase proteins or fragments thereof and one or more amino acid sequences comprising at least one bacterial protease cleavage site, the amino acid sequence comprising:
- Ala-Gly-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Pro-Gly-Gly
- (i.e., SEQ ID NO:
- 1).
A method for detecting the presence of one or more bacterial proteases in a sample comprising:
- exposing the sample putatively containing the one or more bacterial proteases to an enrichment matrix comprising a bioluminescent protease substrate that is capable of eliciting a detectable luminogenic signal when modified by the one or more bacterial proteases, the bioluminescent protease substrate comprising one or more luciferase proteins or fragments thereof and one or more amino acid sequences comprising at least one bacterial protease cleavage site, the amino acid sequence comprising:
- Ala-Gly-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Pro-Gly-Gly
- (i.e., SEQ ID NO:
- 1);
- measuring the level of change in the detectable luminogenic signal;
- and
- detecting the presence of the one or more bacterial proteases when the level of change in the detectable luminogenic signal in the sample is elevated.
A bacterial protease detection kit comprising:
- an enrichment matrix comprising one or more bioluminescent protease substrates comprising one or more luciferase proteins or fragments thereof and one or more amino acid sequences comprising at least one bacterial protease cleavage site, the amino acid sequence comprising:
- Ala-Gly-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Pro-Gly-Gly
- (i.e., SEQ ID NO:
- 1),
- wherein a detectable fluorescence signal is produced upon interaction of the substrate with the bacterial protease.
Dependencies are inferred from the claim text.