Native luciferase

Manufactured by Promega

Native luciferase is a naturally occurring enzyme that catalyzes a bioluminescent reaction. It is responsible for the light-emitting properties observed in certain organisms, such as fireflies. This enzyme can be used in various scientific applications requiring bioluminescent detection or quantification.

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Lab products found in correlation

Sirius luminometer, by Berthold Technologies (1 mentions) Spectramax m5 plate reader, by Molecular Devices (1 mentions) Steadyglo reagent, by Promega (1 mentions)

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Native firefly luciferase (3 citations)

2 protocols using native luciferase

Refolding of Denatured Luciferase

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Luciferase (1.5 μM) was denatured at 44°C for 10 min in buffer D in the presence or absence of 10 μM sHsps as indicated (3 μM IbpA_Ec/Cn and 7 μM IbpB_Ec/Cn in case of IbpAB_Ec/Cn). Protein refolding was started by 40-fold dilution of denatured luciferase in the Hsp70-Hsp100 chaperone cocktail. Unless noted otherwise, the chaperone concentrations used were as follows: Limiting Hsp70—DnaK 0.7 μM, DnaJ 0.28 μM, GrpE 0.21 μM, ClpB 2 μM; saturating Hsp70—DnaK 3.5 μM, DnaJ 1.4 μM, GrpE 1.05 μM, ClpB 2 μM. All assays were performed in the presence of an ATP-regenerating system (18 mM creatine phosphate, 0.1 mg/ml creatine kinase, 5 mM ATP). The disaggregation reaction was carried out at 25°C. Luciferase activity was measured at time points using a Sirius Luminometer (Berthold), normalized and presented as a mean ±SD from at least 3 independent experiments. 100% activity is defined as the maximal activity obtained during the refolding of luciferase-IbpAB assemblies. The normalisation procedure was performed in this way since the native luciferase (Promega) contains the fraction of inactive protein and the activity of refolded luciferase often exceeded the activity of native luciferase preparation used for denaturation (by approximately 20–30%).

Obuchowski I., Piróg A., Stolarska M., Tomiczek B, & Liberek K. (2019). Duplicate divergence of two bacterial small heat shock proteins reduces the demand for Hsp70 in refolding of substrates. PLoS Genetics, 15(10), e1008479.

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Luciferase Refolding Assay with Chaperones

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Experiments were performed as described previously (50 (link)). Briefly, working stocks of denatured luciferase were prepared by mixing 10 μL of 200 μM native luciferase (Promega) with 30 μL of 8 M GnHCl for 1 hour at room temperature. Denatured luciferase stocks were stored at −80 °C until use. To white 96-well plates, was added denatured luciferase (final concentration of 100 nM), Hsp72 or Hsp72ΔEEVD (final concentration of 1 µM), and various concentrations of E. coli DnaJ or human DnaJC7 to give a final volume of 25 μL in refolding buffer (20 mM HEPES, 120 mM KAc, 1.2 mM MgAc, 15 mM DTT, 60 mM creatine phosphate, 35 U/mL creatine kinase, 5 ng/μL BSA, pH 7.4). The reaction was initiated by adding 10 μL of 2.5 mM ATP to give a final concentration of 1 mM. Plates were covered and incubated at 37 °C for 1 hour. Next, 25 μL of Steady-Glo reagent (Promega) was added to each well and luminescence values were measured immediately using a Molecular Devices Spectramax M5 plate reader (Sunnyvale, CA).

Assimon V.A., Southworth D.R, & Gestwicki J.E. (2015). Specific binding of tetratricopeptide repeat (TPR) proteins to heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) is regulated by affinity and phosphorylation. Biochemistry, 54(48), 7120-7131.

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