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Fluorescent Protein Expression Vectors: See Your Data In Color
Buying Tips
Nov 13 '07
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Introduction |
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| The ability to make virtually any protein of interest fluorescent is so common now that we almost take it for granted. The past few years saw an expansion in the variety and availability of fluorescent tags for expressed proteins. Today these are used to label proteins in fixed or live tissues, to label cells by type, and even to label subcellular structures. Fluorescent proteins are also used to track promoter activity, generate cell lines, and label cells and organs in whole body in vivo imaging, among other uses. There are many factors involved in choosing what type of fluorescent tag to use for your protein, and there are so many offerings that you are sure to find what you need. Here are some considerations that will help you begin. |
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What color do you want? |
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| The color, of course, is determined by the fluorophore’s emission wavelength. The rainbow of colors available is not just for aesthetic purposes—choosing the right excitation and emission wavelengths may be important for several reasons. If you are planning multiplex experiments that use more than one fluorophore, you need to make sure that the respective wavelengths of the two fluorophores don’t overlap. On the other hand, this principle may be exploited in FRET (fluorescence resonance energy transfer) experiments, in which the emission wavelength of one fluorophore is very close to the excitation wavelength of another, such that exciting the first one with light results in the activation of the second one, too, provided the two labeled proteins are in close enough physical proximity. Another consideration for choosing wavelength is your system. Most in vitro experiments can support a range of wavelengths, but for in vivo applications such as whole body imaging, only the longer wavelengths in the far red region can penetrate sufficiently through body tissues to be effective.
You can find a spectrum of edible-sounding colors at Clontech, which now distributes some of the fluorescent proteins named for fruits, originally developed in the lab of Roger Tsien at the University of California, San Diego. “We currently offer mCherry, which has already been used widely in many applications, as well as mRaspberry, mPlum, mBanana, mOrange, and mStrawberry,” says Suvarna Gandlur, product manager for fluorescent proteins at Clontech. “The fruit fluorescent proteins comprise a broad range of spectra and increase the multiplexing possibilities of end users. Clontech also provides antibodies for detecting and validating these proteins.”
Evrogen offers a wide collection of fluorescent proteins ranging from cyan to red, in two types. The Turbo line includes bright green, yellow, red/orange, true red, and far-red proteins (their recent far-red product is called Katushka, or TurboFP635) with fast maturation for early detection. Evrogen recommends the Turbo line wherever you need fast appearance of bright signal, such as when labeling cells or organelles, and tracking promoter activity. Evrogen’s Tag line, on the other hand, is more suited to making fusion proteins, such as with protein localization studies and the generation of stable cell lines in long-term cultures. The Tag fluorophores are offered in cyan, green, yellow, red/orange, and far-red (their recent far-red product is called mKate, or TagFP635).
If you’re not sure which fluorophores you might need, consider Promega’s unusual HaloTag system. “HaloTag Technology provides new options for rapid, site-specific labeling of proteins in live or fixed cells and on solid supports,” says Paula Phenix, global product manger in protein analysis at Promega. The technology is based on the efficient formation of a covalent bond between a protein fusion tag (called a HaloTag) and specific interchangeable synthetic ligands, including fluorescent ligands for cellular imaging applications.
“The main challenge for researchers working with GFP (green fluorescent protein) is that researchers have to make multiple protein fusion constructs in order to use more than one fluorophore,” says Phenix. “With the HaloTag Technology, they only have to make one protein fusion construct. Fluorophores can be interchanged among a variety of standard dyes, without requiring changes to the underlying genetic construct. With this one construct, they are able to use many different fluorophores.”
In a similar vein, Covalys Biosciences uses a multifunctional protein tag called SNAP-tag, which is not fluorescent by itself, but more than 10 fluorescent labels can attach to it via a covalent bond. “Covalys now offers SNAP-express pSEMS-Gateway® plasmids for easy and efficient cloning of N- or C-terminal SNAP-tag fusion proteins, followed by transient or stable expression of SNAP-tag fusion proteins in mammalian cells,” says Andreas Brecht, CEO of Covalys. “The SNAP-tag allows a wide range of applications from a single expression, transfection, or most importantly a stable cell line. This shifts the focus away from the cloning and control experiments accompanying each re-cloning step, and back to doing the real work.” |
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What do you want to label? |
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| The type of fluorescent label you choose depends in part on what it is that you want to label. The two types of fluorophores described above from Evrogen illustrates a consideration—that of fluorophore size. The dimeric Turbo line of colors is more suited for labeling cells and organelles, and tracking promoter activity, for example; the smaller monomeric Tag line, in contrast, is better for labeling proteins (via fusions) for protein localization studies and the generation of stable cell lines in long term culture. Evrogen’s products are also sold by another company called BioCat.
If you want to use fluorescent labels to study protein-protein interactions, consider the new kit from MBL International. The latest additions to their fluorescent protein expression vector palette are the CoralHue Fluo-chase Kit, and the CoralHue Keima-Red fluorescent proteins. “The CoralHue Fluo-chase Kit is the world's first fluorescent protein kit that enables the visualization of protein-protein interactions as fluorescent signals in mammalian cells within 24 hours,” says Ricky Kim, marketing manager at MBL International. “The CoralHue Fluo-chase Kit allows for a high sensitivity analysis, especially in weak protein-protein interactions. Unlike any other bimolecular fluorescent [tag], you will be able to see the protein interaction [in the] in vivo state. CoralHue Monomeric and Dimeric Keima-Red have bright red fluorescence with the largest commercially available Stokes shift, making Keima-Red a superb reporter protein for multicolor fluorescence analyses. It is particularly useful when performing dual color fluorescence cross-correlation spectroscopy (FCCS).”
BD Biosciences Pharmingen recently released a collection of eight vectors designed to label specific organelles, and “have been validated in both live and fixed mammalian cell imaging applications,” according to Mary Haldeman, product manager of bioimaging systems at BD Biosciences. “The collection of eight vectors expresses fusion proteins composed of Green or Red FPs (Fluorescent Proteins) and proteins that target various cellular structures or organelles.” The FP vector collection consists of the red FP vectors aimed at the nucleus, mitochondrion, endoplasmic reticulum, and peroxisome, and the green FP vectors, aimed at the nucleus, mitochondrion, actin, and Golgi. “They are organelle location specific vectors—one thing you need to control is the amount of expression, another is transfection efficiency,” says Haldeman. “These things are controlled by other products used for the transient transfection.” |
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Do you want to make your own fusion protein? |
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| Clontech and Invitrogen/Molecular Probes offer two options for those who don’t want to make their own fluorescently labeled protein. The Clontech system works on the principle of co-expression. “The Retro-X™ IRES Living Colors® Vectors are bicistronic vectors designed to coexpress your gene of interest and our brightest red (DsRed-Express) or green (ZsGreen1) fluorescent proteins,” explains Gandlur. “Each protein is translated independently, but from the same promoter, enabling you to determine transfection/transduction efficiency, approximate expression levels for your protein of interest, and enrich for cells expressing your gene of interest via flow cytometry.” Thus the fluorescent protein acts as a reporter for transcription and translation of your protein of interest, without having to use fusion proteins. “This is a big advantage to end users who want to use fluorescent proteins only as reporters or as a measure of their protein without altering their own protein’s structure or function,” says Gandlur.
For another virtually care-free option, Invitrogen’s Organelle Lights™ fluorescent proteins are designed to simplify the construction of fluorescent protein expression vectors. They are ready-to-use fluorescent protein constructs fused with signal peptides that direct them to the targeted subcellular compartments, such as nuclear and plasma membrane, endoplasmic reticulum, Golgi, and peroxisomes. Transfection is mediated by the baculovirus insect virus, with no preparation necessary other than adding the reagent to your cells. The Organelle Lights markers are stable in frozen storage, yet are also suitable for live cell experiments and high content screening applications.
For those who do want to incorporate their protein of interest into a fusion protein to make it fluorescent, Invitrogen/Molecular Probes offers the Vivid Colors™ Fluorescent Protein Gateway® Destination Vectors. These help you to fuse the carboxyl or amino end of your protein to the emerald green fluorescent protein (EmGFP) or yellow fluorescent protein (YFP). Another fusion system is offered by Clontech with their Lenti-X™ Living Colors® Vectors. “The Lenti-X system was designed specifically to deliver your gene of interest into difficult-to-transfect cells, with high efficiency and enhanced safety,” says Gandlur. “Express your gene of interest, fused at the N- or C-terminus to either our monomeric red (DsRed-Monomer) or monomeric green (AcGFP1) fluorescent proteins. The newly designed Lenti-X vectors allow higher levels of protein expression, making fluorescent reporters more effective.”
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Fluorescent proteins of the future |
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| Though the choices of fluorescent protein expression vectors might seem to be burgeoning, scientists still want more. Gandlur expects to see “a wider spectral range of monomeric fluorescent proteins,” and also believes that we could benefit from “developing fluorescent proteins that are further red-shifted (above 650 nm), brighter, and as small as possible without leaving the chromophore unprotected.”
Brecht of Covalys sees that “scientists are demanding an increasing range of fluorescent labels with limited overlap for high content studies in living cells.” He also notes that, “while non cell permeable labels are available up to 780 nm excitation, so far cell permeable high performance labels are available up to 550 nm excitation. Covalys is working on extending the range for cell permeable labels up to and above 600 nm excitation wavelength.”
With these considerations in mind, and any others of your own, the right fluorescent protein expression vector is likely to become apparent soon—in living color. |
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Caitlin Smith
Contributing Writer
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