Scientists at Carnegie Mellon University have developed fluorogen-activated proteins that they say can be used to monitor the biological activities of individual proteins and other biomolecules on the surface of living cells in real time.
The FAPs, which could become a component of molecular biosensor technology being developed at Carnegie Mellon, are described in a paper published in the February issue of Nature Biotechnology.
“The concept that we are working on right now is combining environmentally sensitive fluorescent dyes with engineered proteins that will bind those dyes, and together, they will act as sensors for the biological processes,” said Alan Waggoner, a professor of biological sciences at CMU and the director of the Molecular Biosensor and Imaging Center.
He said that the first platform for doing this is the single-chain antibody (scFv) that MBIC research scientist Christopher Szent-Gyorgyi has been developing over the past several years.
“The bottom line of this paper is that we have been able to select single-chain antibodies from a yeast library that bind several dye molecules [thiazole orange and malachite green] that are non-fluorescent in water or buffer, but that become highly fluorescent when bound to the scFv antibody,” Waggoner said.
In the paper, the investigators showed that they could use this antibody as a label to tag other proteins, for example membrane proteins, and when one of the dyes that are impermeant to the cell is added, then it labels only the protein that is on the surface of the cell.
“We think that this will be a very useful tool for quantifying the amount of a particular protein on the surface of the cell — for example, a transport protein like glucose transport protein — without the complications of also seeing what is in the internal cellular compartments and vesicles,” Waggoner told CBA News this week.
He explained that by contrast, if green fluorescent proteins are used to fuse to the glucose transporter, what is on the surface membrane would be visible, but even larger amounts of fluorescence from internal stores and vesicles would also be visible.
"This will be a very useful tool for quantifying the amount of a particular protein on the surface of the cell … without the complications of also seeing what is in the internal cellular compartments and vesicles.”
Waggoner pointed out that the surface density of glucose transporter on the surface of the cell is under the control of insulin and brings glucose into the cell. “If you want to study diabetes, for example, you would be very interested in the regulatory events that control how much of a transporter is on the surface of the membrane,” he said.
The CMU team believes that this probe, the scFv with the thiazole orange or malachite green, might be an appropriate way of quantifying the surface density of proteins of interest, said Waggoner.
He mentioned that no pharmaceutical companies are currently using this assay, but that the CMU scientists would like to see if the pharma companies might find it useful. Waggoner mentioned that he and his team plan to try to commercialize this technology in the future, or transfer it from the academic environment. He did not offer a timeline for commercialization, however.
In terms of drug discovery, “If you wanted to do high-content cell-based assays for the presence of a receptor on the surface of a cellular membrane, and add different compounds to see if they alter the surface density of that receptor, then this may prove to be a useful and easy assay to do,” Waggoner said.
He added by saying that the investigators want to make sure that the FAPs are a robust technology that is easy to use. “But we will probably work with some partners, so that they can give us feedback. We will probably also try some other surface membrane proteins to make sure that it is a versatile technology.”
The scientists are trying to develop another type of application at the MBIC, which is the work of Peter Berget, Waggoner said. He said that Berget is attempting to develop protease sensors with the FAP technology.
“Live-cell imaging and our understanding of biological processes have been revolutionized by the development of fluorescent probes over the past 15 years,” Sally Kim, a post-doctoral fellow in the division of biology at the California Institute of Technology, wrote in an e-mail to CBA News this week.
She said that the continued development of new fluorescent tools increases the number of options that scientists have in their arsenal to approach a variety of biological questions.
The availability of these tools allows for the optimal selection of the best properties for answering the question at hand, Kim said.
At least one vendor has noticed that tools for live cell imaging could be useful, and has in fact already noticed a shift in the marketplace. George Hanson, a senior research scientist at Invitrogen, said the firm’s Molecular Probes business was seeing that its customers “used to be very much focused on just the content of labeling, in terms of, ‘What could they label in the cell?’ But we have now moved from looking at fixed cells to looking at live cells,” said Hanson. “Our customers are interested in what is happening in a very physiologically relevant state.”
The new trend is delivering molecular probes or fluorophores to live cells without disrupting the normal, physiological process of these cells, Hanson said. “That has been the biggest challenge.”