Information Page for RLuc8, RLuc8.6-535, RLuc7-521, and other variants of RLuc

Background

During the course of my research, I have developed several variants of Renilla luciferase (RLuc) optimized for different applications through a combination of consensus mutagenesis, site-directed mutagenesis, and random-mutagenesis. Those of most use for outside researchers are:

• RLuc8: This variant was selected for enhanced stability in murine serum as well as enhanced enzymatic activity. It is ~4x brighter than RLuc and ~200x more stable than RLuc in murine serum at 37° C. This variant also exhibits increased stability compared to the native enzyme under a variety of additional conditions, including mammalian cytoplasm, lyophilization, and commonly used buffers (e.g. PBS).


• RLuc/M185V/Q235A: This variant was selected for decreased stability in murine serum as well as enhanced enzymatic activity. It has ~50% of the stability of RLuc in murine serum at 37° C while being ~4x brighter than RLuc. In mammalian transfection conditions, this variant retains its ~50% decrease in stability as well as its increase in enzymatic activity.


• RLuc8.6-535 was selected for a red-shifted emission spectrum. It retains the increased stability and enhanced enzymatic activity of RLuc8, and also exhibits an emission spectrum that is red-shifted ~50 nm compared to RLuc.


• RLuc7-521 was selected for a red-shifted emission spectrum without a stabilized phenotype. It retains the same intracellular stability of RLuc while exhibiting an emission spectrum that is ~40 nm red-shifted compared to RLuc.

Obtaining

Please send an email to my former advisor, Dr. Sanjiv Gambhir (sgambhir@stanford.edu), requesting the desired plasmid. As a courtesy, please describe the project in which you intend to use the luciferase variant.

This should get the process started. Stanford requires a Material Transfer Agreement (MTA) prior to shipping out anything, someone from the lab should email you in response regarding how to complete this requirement.

I no longer work in the lab, and as such cannot direct assist this process. However, feel free to email me (Andreas Loening) at loening@stanford.edu if there are any difficulties or any questions

Plasmids

Any plasmid from the references listed below can be requested. The most commonly requested plasmids are:

pBAD-RLuc8 - A bacterial expression plasmid containing RLuc8 intended for use in cytoplasmic expression. A 6xHis tag is present on the C-terminus. If your aim is to produce large amounts of RLuc8 protein in bacteria, this is what you want. [Plasmid Sequence] [Protein Sequence]

pBAD-pelB-RLuc8 - A bacterial expression plasmid containing RLuc8 intended for periplasmic expression. A pelB leader sequence has been fused to the N-terminus of the luciferase, and a 6xHis tag is present on the C-terminus. Yields are about 10-fold less with this construct than the cytoplasmic construct, but the resulting protein will have >95% purity following a single step of nickel affinity chromatography. [Plasmid Sequence] [Protein Sequence]

pBAD-pelB-RLuc (with Myc epitope) - Some of the initial pBAD-pelB constructs contained a Myc epitope between the c-terminus of the luciferase and the 6xHis tag. This Myc epitope was removed during the construction of the pBAD-pelB-RLuc8 plasmid. [Plasmid Sequence]

pcDNA-RLuc8 - A mammalian expression plasmid containing RLuc8. The backbone is pcDNA 3.1, and no 6xHis tag is present. If you want to use RLuc8 as a reporter gene in experiments using mammalian cells, this is the one for you. [Plasmid Sequence] [Protein Sequence]

pcDNA-RLuc8.6-535 - A mammalian expression plasmid containing RLuc8.6-535. The backbone is pcDNA 3.1, and no 6xHis tag is present. If you want to use RLuc8.6-535 as a reporter gene in experiments using mammalian cells, this is what you want. [Plasmid Sequence] [Protein Sequence]

pcDNA-RLuc7-521 - A mammalian expression plasmid containing RLuc7-521. The backbone is pcDNA 3.1, and no 6xHis tag is present. If you want to use RLuc7-521 as a reporter gene in experiments using mammalian cells, this is what you want. [Plasmid Sequence] [Protein Sequence]

References

The paper describing the development and characterization of stabilized, destabilized, and enzymatically enhanced variants of RLuc (e.g. RLuc8) is:

Loening AM, Fenn TD, Wu AM, Gambhir SS
Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output
Protein Engineering, Design and Selection 19:391-400, 2006. [Manuscript] [Postprint PDF]

A paper that includes a detailed protocol on producing and purifying RLuc8 cytoplasmically is:

So M-K, Loening AM, Gambhir SS, Rao J
Creating self-illuminating quantum dot conjugates
Nature Protocols 1:1160-1164, 2006. [Manuscript][Direct Link]

The paper described the development of red-shifted variants of RLuc8 (e.g.RLuc8.6-535) is:

Loening AM, Wu AM, Gambhir SS
Red-shifted Renilla reniformis luciferase variants for imaging in living subjects
Nature Methods 4(8):641-643, 2007. [Manuscript] [Preprint PDF]

The paper described the development of the red-shifted variant of RLuc (RLuc7-521) for transient reporter gene imaging is:

Loening AM, Dragulescu-Andrasi A, Gambhir SS
A red-shifted Renilla luciferase for transient reporter-gene imaging
Nature Methods 7(1):5-6, 2010. [Manuscript]

Structures

The structure for RLuc8 has been solved by x-ray crystallography. PDB files of the structure are available from the RCSB PDB with the ID's of 2PSES, 2PSD, 2PSH, 2PSJ, and 2PSF. The structure most likely to be useful to you is 2PSD.

The paper describing these structures is:

Loening AM, Fenn TD, Gambhir SS
Crystal structures of the luciferase and green fluorescent protein from Renilla reniformis
J. Mol. Biol., 374(4):1017-1028, 2007. [Manuscript] [Postprint PDF]

Questions