1. Cloning, Over-expression and Purification of NanoLuc Luciferase
Dr. Angie Spencer and Holly DuPlain

2. Resonance Energy Transfer
Energy transfer from donor to acceptor molecule
Only possible within 5 nm – 10 nm
Protein-protein interactions
Inhibitors
Protein-protein interactions (PPI) are important because they can lead to the development of drugs that target those interactions. One useful way to study PPI is a process called resonance energy transfer. One fluorophore is fused to one of the interacting proteins and another fluorophore is fused to that protein’s interaction partner. As one of the fluorophores (the donor) is excited via an external source or via chemical reaction, energy is transferred to the other fluorophore (the acceptor) if these two molecules are close enough and absorption and emission spectra overlap. Consequently, if two proteins are interacting, this can be shown when the donor is excited and then the acceptor is excited. Additionally, if a RET signal is being produced but disappears with the addition of a third molecule it suggests the third molecule may inhibit the original interaction. RET technology comes in two forms: Fluorescent Resonance Energy Transfer (FRET) and Bioluminescence Resonance Energy Transfer (BRET).

3. Fluorescence Resonance Energy Transfer (FRET)
Fluorescent proteins
Excitation from external source
Phototoxicity, photobleaching
Excitation of acceptor
High expression levels
FRET requires an outside light source. A laser is commonly employed. Proteins commonly used are autofluorescent (YFP, GFP, etc.) While FRET is useful, there are problems associated with it including the need for high expression levels of the studied proteins which may not reflect physiological conditions. Photoxicity is possible in addition to photobleaching, meaning either the cells will die or the fluorophores will not longer be active. Additionally, it is possible to excite the acceptor from the external light source without exciting the donor first. Finally, background fluorescence can occur. The other method utilizing resonance energy transfer known as BRET solves many of these problems.
PhiYFP

4. Bioluminescence Resonance Energy Transfer (BRET)
Donor is capable of bioluminescence
Chemical reaction produces light
Rluc, Fluc and NanoLuc

5. Bioluminescence Resonance Energy Transfer (BRET)
Machleidt, T., et al. ACS Chem. Biol. 2015, 10,

6. Bioluminescence Resonance Energy Transfer (BRET)
NanoLuc + ? = Optimized BRET

7. Research Scheme Subcloning Transforming Over-expressing Purification
Tagging

8. Research Scheme - Cloning
Nanoluc Gene (Nluc)
pET21 Plasmid
pET21Nluc
Restriction Enzymes
Ligase
PCR
Restriction Enzyme Cleavage Site
Restriction Enzyme Cleavage Site

9. Vector Map Amp Resistance Lac Repressor C-terminal His Tag
Novagen, 1999

10. Research Scheme - Cloning_
Xho I
Nde I
Both
Ladder
Empty
10.0 kb
Screening
3.0 kb
Empty Plasmid: 5.4 kb
Nanoluc Gene: 0.5 kb
1.5 kb
1.0 kb
0.5 kb
Transformed E.coli BL21(DE3) on Ampicillin Plates
1% Agarose

11. Research Scheme – Expression and Purification
IPTG
Sonication
Affinity Chromatography
Taken from Qiagen Manual

12. SDS-PAGE of Purified NanoLuc
Sample
Ladder
205,748 Da
79,695 Da
34,107 Da
Molecular Weight NanoLuc: 19,000 Da
17,559 Da
6,088 Da
10% Acrylamide

13. Activity Assay Results
100 ng/µL
0.0 ng/μL
Increasing Concentration of NanoLuc
Inouye, S. & Satoko, S., Protein Purification and Expression. 2007, 15,
Reaction Buffer (30 mM Tris-HCl, pH 7.6, 10 mM EDTA)

14. Bioluminescence Resonance Energy Transfer (BRET)
NanoLuc + ? = Optimized BRET

15. Tagging with Alexa Fluor 488
NanoLuc emission max = 454 nm
Alexa Fluor 488 excitation max= 494 nm
Alexa Fluor 488 emission max = 517 nm

16. Summary Successfully cloned, over-expressed and purified NanoLuc
Presence of active NanoLuc confirmed by activity assay
Tagged NanoLuc with Alexa Fluor 488
Tag purified NanoLuc with various fluorophores

17. Summary Machleidt, T., et al. ACS Chem. Biol. 2015, 10, 1997-1804

18. Acknowledgements
Dr. Nevin Lambert, GRU Department of Pharmacology and Toxicology
Dr. Angie Spencer
GRU Department of Chemistry and Physics
Center for Undergraduate Research and Scholarship

19. Cloning, Over-expression and Purification of NanoLuc Luciferase
Dr. Angie Spencer and Holly DuPlain