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A novel bioluminescent protease assay using engineered firefly luciferase.

Wigdal SS, Anderson JL, Vidugiris GJ, Shultz J, Wood KV, Fan F - Curr Chem Genomics (2008)

Bottom Line: Understanding their biological functions underpins the efforts of drug discovery.Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold.The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased.

View Article: PubMed Central - PubMed

Affiliation: Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA.

ABSTRACT
Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening.

No MeSH data available.


Related in: MedlinePlus

Firefly luciferase is a 61 kDa monomeric enzyme that catalyzes the oxidation of firefly luciferin in the presence of ATP and oxygen to emit yellow-green light. Upon binding of substrates, the structure of firefly luciferase undergoes conformational changes from open to closed forms. We created a circularly permuted luciferase by covalently joining the native N and C termini of firefly luciferase through the cloning in of a short polypeptide linker containing a protease recognition sequence. This results in restricting the movement between the two domains and locking the enzyme in the less active open form. Protease cleavage releases this constriction thereby restoring higher activity. A. To express this mutant luciferase, new N and C termini were inserted at amino acids 234 and 233, respectively. B. Insertion of the polypeptide linker greatly reduces luciferase activity. Proteolytic cleavage by the cognate protease (scissors) activates the mutant luciferase enzyme resulting in a luminescent signal in the presence of the luciferin substrate (yellow circle).
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Figure 1: Firefly luciferase is a 61 kDa monomeric enzyme that catalyzes the oxidation of firefly luciferin in the presence of ATP and oxygen to emit yellow-green light. Upon binding of substrates, the structure of firefly luciferase undergoes conformational changes from open to closed forms. We created a circularly permuted luciferase by covalently joining the native N and C termini of firefly luciferase through the cloning in of a short polypeptide linker containing a protease recognition sequence. This results in restricting the movement between the two domains and locking the enzyme in the less active open form. Protease cleavage releases this constriction thereby restoring higher activity. A. To express this mutant luciferase, new N and C termini were inserted at amino acids 234 and 233, respectively. B. Insertion of the polypeptide linker greatly reduces luciferase activity. Proteolytic cleavage by the cognate protease (scissors) activates the mutant luciferase enzyme resulting in a luminescent signal in the presence of the luciferin substrate (yellow circle).

Mentions: We have previously described a novel biosensor using a genetically modified firefly luciferase that allows the facile interrogation of protease function without chemical synthesis [12]. It uses a bioluminescent substrate generated through molecular cloning and transcription/translation coupled cell-free expression. Thus protease substrates do not need to be purchased or chemically synthesized. The modified firefly luciferase is covalently joined at the native termini with a short peptide containing the protease recognition site which serves to restrict the luminescent reaction. Proteolytic cleavage of the peptide by the cognate protease activates the luciferase enzyme, typically over 100 fold. To express this mutant luciferase, new termini were inserted to create the circularly permuted form of firefly luciferase. The design strategy of this assay is shown in Fig. (1). Importantly, we have shown that this mutant luciferase protease assay retains the advantages of the bioluminescent format, which include increased sensitivity and wide dynamic range, while accommodating proteases with and without P' requirements [12].


A novel bioluminescent protease assay using engineered firefly luciferase.

Wigdal SS, Anderson JL, Vidugiris GJ, Shultz J, Wood KV, Fan F - Curr Chem Genomics (2008)

Firefly luciferase is a 61 kDa monomeric enzyme that catalyzes the oxidation of firefly luciferin in the presence of ATP and oxygen to emit yellow-green light. Upon binding of substrates, the structure of firefly luciferase undergoes conformational changes from open to closed forms. We created a circularly permuted luciferase by covalently joining the native N and C termini of firefly luciferase through the cloning in of a short polypeptide linker containing a protease recognition sequence. This results in restricting the movement between the two domains and locking the enzyme in the less active open form. Protease cleavage releases this constriction thereby restoring higher activity. A. To express this mutant luciferase, new N and C termini were inserted at amino acids 234 and 233, respectively. B. Insertion of the polypeptide linker greatly reduces luciferase activity. Proteolytic cleavage by the cognate protease (scissors) activates the mutant luciferase enzyme resulting in a luminescent signal in the presence of the luciferin substrate (yellow circle).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 1: Firefly luciferase is a 61 kDa monomeric enzyme that catalyzes the oxidation of firefly luciferin in the presence of ATP and oxygen to emit yellow-green light. Upon binding of substrates, the structure of firefly luciferase undergoes conformational changes from open to closed forms. We created a circularly permuted luciferase by covalently joining the native N and C termini of firefly luciferase through the cloning in of a short polypeptide linker containing a protease recognition sequence. This results in restricting the movement between the two domains and locking the enzyme in the less active open form. Protease cleavage releases this constriction thereby restoring higher activity. A. To express this mutant luciferase, new N and C termini were inserted at amino acids 234 and 233, respectively. B. Insertion of the polypeptide linker greatly reduces luciferase activity. Proteolytic cleavage by the cognate protease (scissors) activates the mutant luciferase enzyme resulting in a luminescent signal in the presence of the luciferin substrate (yellow circle).
Mentions: We have previously described a novel biosensor using a genetically modified firefly luciferase that allows the facile interrogation of protease function without chemical synthesis [12]. It uses a bioluminescent substrate generated through molecular cloning and transcription/translation coupled cell-free expression. Thus protease substrates do not need to be purchased or chemically synthesized. The modified firefly luciferase is covalently joined at the native termini with a short peptide containing the protease recognition site which serves to restrict the luminescent reaction. Proteolytic cleavage of the peptide by the cognate protease activates the luciferase enzyme, typically over 100 fold. To express this mutant luciferase, new termini were inserted to create the circularly permuted form of firefly luciferase. The design strategy of this assay is shown in Fig. (1). Importantly, we have shown that this mutant luciferase protease assay retains the advantages of the bioluminescent format, which include increased sensitivity and wide dynamic range, while accommodating proteases with and without P' requirements [12].

Bottom Line: Understanding their biological functions underpins the efforts of drug discovery.Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold.The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased.

View Article: PubMed Central - PubMed

Affiliation: Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA.

ABSTRACT
Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening.

No MeSH data available.


Related in: MedlinePlus