Cloning, Over-expression and Purification of NanoLuc Luciferase Dr. Angie Spencer and Holly DuPlain http://www.promega.com/products/pm/nanoluc-luciferase-redefining-reporter-assays/

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). http://www.biotek.pt/pt/resources/articles/fluorescent-proteins-filters-mirrors-wavelengths.html

Fluorescence Resonance Energy Transfer (FRET) Fluorescent proteins Excitation from external source Phototoxicity, photobleaching Excitation of acceptor High expression levels http://www.evrogen.com/products/PhiYFP/PhiYFP_Detailed_description.shtml 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

Bioluminescence Resonance Energy Transfer (BRET) Donor is capable of bioluminescence Chemical reaction produces light Rluc, Fluc and NanoLuc http://www.reportergene.com/2013/01/the-nanoluc-luciferase-reporter-dossier.html

Bioluminescence Resonance Energy Transfer (BRET) Machleidt, T., et al. ACS Chem. Biol. 2015, 10, 1997-1804 http://www.biotek.pt/pt/resources/articles/fluorescent-proteins-filters-mirrors-wavelengths.html

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

Research Scheme Subcloning Transforming Over-expressing Purification Tagging http://www.reportergene.com/2013/01/the-nanoluc-luciferase-reporter-dossier.html

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

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

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

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

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

Activity Assay Results 100 ng/µL 0.0 ng/μL Increasing Concentration of NanoLuc Inouye, S. & Satoko, S., Protein Purification and Expression. 2007, 15, 261-268 Reaction Buffer (30 mM Tris-HCl, pH 7.6, 10 mM EDTA)

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

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

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

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

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

Cloning, Over-expression and Purification of NanoLuc Luciferase Dr. Angie Spencer and Holly DuPlain http://www.promega.com/products/pm/nanoluc-luciferase-redefining-reporter-assays/