Takara Bio division Clontech this week said that it has signed a worldwide commercial licensing agreement with Bayer Healthcare that will allow it to sell reporter assay kits based on a Bayer luciferase technology.
The deal is one in a string of recent or planned moves by the former Becton Dickinson unit designed to strengthen its position in the cell-based assay reagent market and fill the hole left by the expiration last year of Clontech’s commercial rights to the Aequorea victoria fluorescent protein cloning vectors.
Under the terms of the Bayer deal, Clontech will be able to sell Ready-to-Glow Secreted Metridia Luciferase Systems directly to academic and commercial accounts for use in high-throughput screening and research. Financial terms were not disclosed.
The assay kits are based on a secreted luciferase from the marine copepod Metridia longa, isolated by scientists from Bayer and the Russian Academy of Sciences in 2003. The cloning and expression of the Metridia luciferase cDNA is described in a January 2004 Journal of Biological Chemistry paper, and later that year Bayer applied for a US Patent protecting the specific isolated luciferases – application number 20040219527 – which is still pending.
Since then Bayer scientists have been optimizing and using the reporter enzyme in their own high-throughput cell-based screening campaigns to monitor gene expression and measure reporter gene activity, Andrew Farmer, director of business development for Clontech Laboratories, told CBA News this week.
“We’ve been looking to expand our technologies in this area for a while,” Farmer said. “We think we have a lot of strengths with fluorescent proteins as reporters. And we’ve been looking to expand that into other areas, and we saw this as an opportunity.
“This brings value that is different both from our fluorescent proteins and more traditional luciferases,” he added. “Bayer has certainly demonstrated that value in its use of this technology in ultra-high-throughput screening in 1,536-well plates on a massive scale, which really shows you the reporter’s sensitivity, and the ability to do this in a real-world setting as pharmaceutical or drug-discovery companies would want to do.”
Luciferase isolated from the firefly is by far the most commonly used luciferase reporter in basic research and drug discovery. Assay kits based on firefly luciferase are sold by a slew of molecular biology reagent vendors.
All luciferase-based reporters work essentially the same way: At its most basic, the enzyme is co-expressed with a gene of interest, and luciferase is produced when that gene is activated. Luciferase production, and therefore gene activity, is monitored by adding a substrate that is converted by luciferase into a detectable luminescent signal.
According to Farmer, the main difference between firefly luciferase and Metridia luciferase is that the former is not a secreted enzyme, while the latter is. Therefore, when using firefly luciferase, scientists must lyse the cell to conduct the detection step.
However, Metridia luciferase is constantly secreted by the cell into the surrounding media, meaning researchers can add the appropriate substrate at any point and detect the luciferase-catalyzed luminescence without harming the cell.
“This gives you a few advantages,” Farmer said. “One, because you’re not killing the cells, you can keep reading this, so you get kinetic data. This is something you can also get from fluorescent proteins, to some extent, but they don’t have the sensitivity range of an enzymatic assay. The kinetic ability is a great advantage over a standard firefly luciferase endpoint assay.”
A second advantage is the ability to “set a clear zero,” Farmer said.
“If you transfect your cells with another reporter, you may get some basal level of expression of that reporter, and then you add your drug and compare [to that],” he said. “Depending on the stability of the protein, there may be something there, but you’re measuring that background.”
With a secreted reporter, however, a researcher can “essentially set the background to time zero by just washing the cells and starting again,” Farmer said. “So even if some basal level of expression was there, if you wash the cells and put fresh medium on, there would be no signal there. If you then add a drug at that point, then everything you get drug-induced.”
Filling the GFP Gap
BD Biosciences sold Clontech Laboratories to Takara Bio for $60 million last year (see CBA News, 7/11/2005). Takara inherited a portfolio of molecular biology regents including cellular reporters, PCR products, inducible RNAi systems, antibodies, and expression vectors, to name a few.
But key legacy products that were missing were cloning vectors for GFP from the jellyfish A. Victoria, one of the most widely used molecular biology tools. The A. victoria proteins had historically been commercially available primarily from BD Biosciences Clontech through a sublicensing agreement from PanVera.
“Bayer has certainly demonstrated that value in its use of this technology in ultra-high-throughput screening in 1,536-well plates on a massive scale.”
However, in 2003 Invitrogen acquired PanVera, and with it the right to commercialize fluorescent proteins based on A. victoria. At that point, Invitrogen and BD Biosciences Clontech both sold the product; but last year, Clontech’s original sublicensing agreement ran out, making Invitrogen the sole supplier of the cloning vectors (see CBA News, 3/15/2005).
At the time, a BD Biosciences official told CBA News that Clontech was already busy developing its own alternative fluorescent and other reporter proteins that “while not as widely accepted in the literature, could provide it with a competitive product line.”
Clontech under Takara has carried on that quest, and the Bayer agreement is an important step.
Farmer said that Takara inherited from BD Biosciences a monomeric red fluorescent protein called DsRed. Red fluorescent proteins are desirable in molecular biology applications because of their distinct emission properties, but have traditionally been limited in their utility as fusion tags because of their tetrameric structure, which causes them to clump.
Though the Ds-Red monomer was previously only available to academic researchers, Clontech will soon make it widely available through a licensing program. Farmer said the Clontech Ds-Red is different from the monomeric red fluorescent protein developed by Roger Tsien at the University of California, San Diego, although he added that “I think you will actually see us taking on the Tsien monomers, as well, in the future.” He declined to elaborate.
Clontech also has the inside track on another fluorescent protein being developed by scientists at the Russian Academy of Sciences and Russian biotech firm Evrogen thanks to a long-standing collaboration between the three entities. These proteins, cloned from the sea coral Dendronephthya, are photo-activated fluorescent proteins that change their emissions from green to red following activation with blue light. This property could be useful in long-term kinetic studies of cellular protein and organelle movement and interactions (see CBA News, 3/29/2006).
Farmer said that cellular assay reagents will “continue to be a strong area of focus for Clontech.”