The Development of mNeonGreen

This week our most recent publication, “A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum” will be published in Nature Methods. It has already been viewable online for some time now, here is a link. We believe this new protein possesses a great deal of potential to advance the imaging fields through enhanced fluorescent microscopy. mNeonGreen enables numerous super resolution imaging techniques and allows for greater clarity and insight into one’s research. As a result of this we are taking a new approach at Allele for distribution of this protein, and here we will describe the history of the protein and some of the factors that led us down this path.

mNeonGreen was developed by Dr. Nathan Shaner at Allele Biotechnology and the Scintillon Institute through the directed evolution of a yellow fluorescent protein we offer called LanYFP. LanYFP is a super bright yellow fluorescent protein derived from the Lancelet fish species, characterized by its very high quantum yield, however, in its native state LanYFP is tetrameric. Dr. Shaner was able to monomerize the protein and enhance a number of beneficial properties such as photostability and maturation time. The result is a protein that performs very well in a number of applications, but is also backwards compatible with and equipment for GFP imaging.

Upon publication there was a question of how distribution should be structured. How would we make this protein available to researchers in a simple manner was a very difficult challenge? We also relied heavily on Dr. Shaner’s knowledge and experience in these matters, as he related his experiences to us from his time in Roger Tsien’s lab at UCSD. When the mFruits was published their lab was inundated with requests. The average waiting period was 3 months to receive a protein and they required a dedicated research technician to handle this process. Eventually the mFruits from the Tsien lab were almost exclusively offered through Clontech. Thus we decided that Allele Biotechnology would handle the protein distribution and take a commercial approach to drastically decrease the turnaround time. The next challenge we faced was how to charge for this protein. Due to the cost of developing this protein, which was fully funded by Allele, there is a necessity to recoup our investment and ideally justify further development of research tools, but we also understand the budget constraints every lab now faces. From this line of thinking we conceived our group licensing model; we wanted to limit the charge to $100 per lab. The way this is fiscally justifiable is having every lab in a department or site license the protein at this charge, including access to all related plasmids made by us as well as those generated by other licensed users (Click here for our licensing page). The benefit we see to this is that the protein is licensed for full use at a low cost, and collaboration amongst one’s colleagues is not only permissible, it’s encouraged. We saw this as a win-win situation. We would recoup our cost and invest in further fluorescent protein research, and our protein costs would not be a barrier to research and innovation.

The granting of a license to use but not distribute material is not unique to commercial sources. Although academic material transfer agreements typically contain specific language forbidding distribution of received material beyond the recipient laboratory, some researchers choose to disregard these provisions. Unfortunately through this action they are disrespecting the intellectual property rights of the original researchers as well as violating the terms of the legal contract they signed in order to receive the material. We believe most researchers choose to respect the great deal of effort that goes into the creation of research tools for biology and do not distribute any material received from other labs without their express permission. However for a company that funds its own basic research our focus is often on the former example rather than the latter. We believe that this focus artificially drives up the costs of licensing a fluorescent protein and obtaining the plasmid, thus we have chosen to believe researchers will respect our intellectual property as long as we are reasonable in our distribution which is something we have truly striven for.

Additionally we believe the broad-range usage of a superior, new generation FP is an opportunity to advocate newer technologies that can be enabled by mNeonGreen, together with a number of Allele’s other fluorescent proteins (such as the photoconvertible mClavGR2, and mMaple). These new imaging technologies are called super resolution imaging (MRI). They provide researchers with a much finer resolution of cellular structures, protein molecule localizations, and protein-protein interaction information. We have started the construction of a dedicated webpage to provide early adopters with practical and simple guidance, click here to visit our super resolution imaging portal.

Tags: cell imaging, fluorescent protein, FP, GFP, mNeonGreen, Superresolution

Allele’s pallet of the super star fluorescent proteins

“Photoblog”–just some fun pictures from our notebooks.

The brightest cyan, green fluorescent proteins, and the brightest ever FP in LanYFP!

Ain't they pretty?

These fluorescent proteins are representatives of the growing family or high quality, new generation FPs engineered to enable experiment previously deemed impossible.

Cells infected with lentivirus carrying mWasabi. Lentivirus carrying LanYFP will make most cells much more brighter than this.

The mWasabi is stimulating

The brightest green fluorescent protein with excellent photostability, carried on 10e8 TU/ml high titer lentivirus.

The LanFPs express well in bacteria.

The LanFPs express well in bacteria

Project planning is under way to test the cytotoxicity of lanFPs in different mammalian cell lines and in vivo with a focus on neurons.

The FPs fold so strongly that they fluorescence even in SDS-PAGE.

Can you see the FP bands in the SDS PAGE?

FPs in SDS PAGE–a closer look

Can you see them now?

FPs in gel cassette over UV lights

Invincible FPs

FPs in gel cassette under blue LED

Fluorescence in SDS page under blue LED

The purified FPs can be used as “real time” protein markers.

New Product of the Week 07/26/10-08/01/10: pCHAC-mWasabi-C for expressing mWasabi fusion through retroviral vectors.

Promotion of the Week 07/26/10-08/01/10: Get 3′ TAMRA & BHQ oligo mods for $45 ea & 3′ Dabcyl mod for $20 50 nmol syn scale only/while supplies last- use dbtkrm0726

Tags: Fluorescent proteins, FP, FPs, FRET, GFP, lab note, lentivirus, mCherry, mTFP1, mWasabi, photos, pictures

Monomeric Photoconvertible Fluorescent Protein for Imaging of Dynamic Protein Localization

Allele Biotech has just made a news announcement indicating that researchers from Dr. Campbell’s lab at the University of Alberta, Canada, and scientists at Allele Biotech Drs. Nathan Shaner and Jiwu Wang published a paper in the Journal of Molecular Biology on July 5th introducing a new photoconvertible fluorescent protein mClavGR.

The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and overcome the limitations imposed by the oligomeric structure of the natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 (ICAM-1) both when the membrane is in contact with a T lymphocyte expressing leukocyte function-associated antigen 1 (LFA-1) and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein sub-populations and subsequent tracking of dynamic changes in localization in living cells.

Compared with previously available photoconvertible FPs, mClavGR2 has much improved photostability of the red state under confocal illumination conditions, 3644 over mEOS2’s 2700 and Dendra2’s 2420. Most notable among other advantages of mClavGR2 is its monomeric structure, its highly optimized and relatively rapid folding efficiency, and its high photoconversion effi ciency due to the high pKa of the green state. Its brightness in both the green and the red states is similar to the popular mCherry.

In regard to monomeric state, the monomeric variant of EosFP, known as mEos, was created through the introduction of two point mutations that disrupted the protein-protein interfaces of the tetrameric species. Expression of mEos at temperatures of greater than 30 °C is problematic, but an effectively monomeric tandem dimer variant does express well at 37 °C. mEos2 has been reported to retain some propensity for dimer formation.

We anticipate that this new addition to the toolbox of engineered FPs will be of great utility in imaging of fast protein dynamics in live cells. Experiments to determine whether the advantages of mClavGR2 translate to improved performance in super-resolution imaging applications have been initiated.

Hiofan Hoi(a), Nathan C. Shaner(b), Michael W. Davidson(c), Christopher W. Cairo(a), d, Jiwu Wang(b) and Robert E. Campbell(a)
a University of Alberta, Department of Chemistry, Edmonton, Alberta, Canada T6G 2G2
b Allele Biotechnology, 9924 Mesa Rim Road, San Diego, California 92121
c National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, 1800 E. Paul Dirac Dr., Tallahassee, Florida 32310
d Alberta Ingenuity Centre for Carbohydrate Science
Received 20 February 2010; revised 15 June 2010; accepted 25 June 2010. Available online 5 July 2010.

New Product of the Week 070510-071110: mClavGR greeen-to-red photoconvertible fluorescent protein, catalogue number to be created

Promotion of the Week 070510-071110: Purified lanYFP, bright even in SDS-PAGE gel WITHOUT dye or excitation, great for in gel marker.

Tags: Dendra2, dynamic imaging, FP, FPs, mCherry, mEOS, mKirGR, PALM, photoconvertible, photoswitchable, STORM

Brightest Ever Fluorescent Protein

LanYFP, identified from lancelet (also known as amphioxus, e.g. Branchiostoma floridae), has been found to have the following properties:

Excitation 513nm
Emission 524nm
Quantum yield 0.95
Extinction coefficient 150,000
pKa ~3.5
Salt insensitive 0-500mM NaCl

LanYFP has a brightness of 143! For comparison, the brightness of the previously known brightest FPs is 95 for tdTomato, and 34 for commonly used EGFP.

Allele already has been exclusively providing the brightest cyan FP in mTFP1 (brightness of 54); and the brightest green FP in mWasabi (brightness of 56). The confirmation of LanYFP as the brightest ever FP is a major milestone of Allele’s research and development efforts in the fluorescent protein field. We are currently monomerizing LanYFP and another lancelet protein, LanRFP. Once completed, the new proteins should definitely be the FPs of choice for in vivo imaging and FRET with unprecedented utilities.

Promotion of the week 062010-061610: Validated Rex1 Promoter Reporter Lentiviral Particles-1 Vial for $149.00 (ABP-SC-RREX2R1). Save $59 if place an order this week! http://www.allelebiotech.com/shopcart/index.php?c=200&sc=34

New product of the week, recombinant mTFP1, mWasabi, LanYFP, LanRFP, $159 for 125 ug, compare price for 100ug vs 125ug in other companies’ offers, you will know that you are getting a good deal from Allele.

Tags: brightest FP, fluorescent protein, Fluorescent proteins, FP, FP brightness, FPs, Lancelet, monomer FP, monomerization, mTFP1, mWasabi, RFP, YFP

mTFP1 is an excellent FRET donor

Because of its excitation and emission wavelength, sharp excitation and emission peaks, high quantum yield, and exceptional photostability, mTFP1 has always been considered a very good Forster resonance energy transfer (FRET) donor (1). More recently, several groups have investigated the use of mTFP1 in various FRET experiments and imaging modalities and have shown that mTFP1 is indeed one of the best choices (2, 3, 4).

In one recent publication, Padilla-Parra et al (2) tested a number of different FRET couples to determine which was the best for fluorescence lifetime imaging (FLIM)-FRET experiments, and found that the mTFP1-EYFP pair was by far the best pair for FLIM-FRET. This group also confirmed that the fluorescence lifetime decay of mTFP1 fits well to a single exponential, and that the time constant for this decay is unaffected by photobleaching, making mTFP1 an excellent choice for any kind of fluorescence lifetime imaging applications, including FLIM-FRET. This group also notes that it is likely that the use of Venus or mCitrine variants in place of EYFP would improve the performance of this FRET pair even further.

In a mathematical analysis of the potential FRET efficiency of mTFP1 with Venus YFP, Day et al. (3) showed that compared with Cerulean (currently the brightest cyan Aequorea GFP variant), one can expect up to 17% better FRET efficiency using mTFP1. This group went on to characterize the mTFP1-Venus pair in live-cell FRET and FLIM-FRET experiments and showed that it worked as predicted in both cases. They also note that mTFP1 has superior brightness and photostability when compared to Cerulean in live cells, which is consistent with all in vitro data reported previously (1). In a related paper, Sun et al. (4) demonstrated that mTFP1 is also an excellent FRET donor for the orange fluorescent protein mKO2.

Together, these recent independent studies confirm that mTFP1 among the best options when choosing a fluorescent protein as a FRET donor. With its proven track record of successful fusions, mTFP1 is also an excellent all-around performer that will enhance almost any live-cell imaging experiment.

(1) Ai et al., (2006) Biochem. J. 400:531-540.
(2) Padilla-Parra et al., (2009) Biophys J. 97(8):2368-76.
(3) Day et al., (2008) J Biomed Opt. 13(3):031203.
(4) Sun et al., (2009) J Biomed Opt. 14(5):054009.

AlleleBlog Admin, by Nathan Shaner

Video of the month (NEW!): Protein Expression Systems on youtube (http://www.youtube.com/watch?v=n81orbUebsQ) and at our protein expression page.

Discount of the week (Dec 14-20): 15% off Phoenix Retrovirus Expression System 2.0 (with selection medium provided)

New product(s) of the week: 48 fluorescent protein fusions on ready-to-infect virus that get into primary mammalian cells as subcellular markers (http://www.allelebiotech.com/shopcart/index.php?c=197&sc=34), 20 infections, only $249 for a limited introduction time.

Tags: cell imaging, CFP, FLIM-FRET, Fluorescent proteins, FP, FRET, FRET acceptor, FRET donor, FRET pair, GFP, GFP fusion, mTFP1, mWasabi, subcellular localization