by Jeffrey M. Perkel
Study any given protein thoroughly enough, and it’s a sure bet that at some point you’ll either need to visualize its intracellular location, monitor its turnover, identify its molecular partners, or characterize its post-translational modifications. For some well-characterized (read: widely investigated) proteins, investigators have available pre-made reagents and kits for performing many if not all of these assays—for instance, using fluorescently labeled antibodies. But suppose your protein is a bit less widely studied. How would you develop the tools to perform the experiments you need?
According to Rizwan Farooqui, market segment manager at Thermo Scientific Pierce Protein Research, there are two fundamental applications for tagging proteins: detection and purification. “Maybe you will detect with Western blotting or immunofluorescence, or use the tag as a fishhook to pull the protein out of cells,” he says.
Fortunately, today’s protein biologists have at their disposal a range of tools for tagging proteins to do just that. Some work in vivo, others in vitro. Many are as easy as a simple one-pot chemical reaction, others require cloning. Whatever your needs, one thing is certain: You never need rely on pre-tagged proteins again. Now you can build your own.
In vivo labeling
The simplest, and perhaps most old-school approach, involves feeding cells isotopically (or otherwise) labeled amino acids or metabolites and letting the cells do the labeling for you. Two classic examples involve growing cells in the presence of [gamma-32P]-labeled ATP (to determine whether, and where, a given protein is phosphorylated) or [35S]-methionine (to label all newly synthesized proteins).
Alternatively, you can feed cells “photoactivatable” amino acids such as Thermo’s L-Photo-Leucine or L-Photo-Methionine (to facilitate cross-linking studies), or even so-called “bio-orthogonal” amino acids. According to Farooqui, “one of the more famous examples” of a bio-orthogonal (or chemoselective) amino acid is one that incorporates an azide group; azide-modified proteins can then be covalently linked in vitro to a molecular tag (such as a fluorophore or biotin) via so-called “Staudinger chemistry,” which couples the tag to the protein via reaction of the azide with a phosphine-containing reagent. (Thermo will begin releasing a line of Staudinger reagents this September.)
SILAC, or “Stable Isotope Labeling with Amino acids in Cell culture,” uses isotopically labeled (with deuterium, 13C and 15N) amino acids (e.g. lysine, arginine) in a cell culture to produce proteins that have the heavy amino acid incorporated, thus causing a mass shift compared to proteins expressed in a normal media.
“SILAC is a metabolic labeling [process],” explains Babu Purkayastha, director of R&D for chemistry and consumables in the Mass Spectrometry Division at Life Technologies, which sells SILAC reagents. “The protein itself is expressed in medium containing isotopically labeled amino acids, and when it is expressed it picks up the heavy amino acid, e.g. lysine or arginine, so now when you isolate that protein, it contains amino acids that are isotopically labeled,” and that can be mass-spectrometrically distinguished from the identical protein grown in “light” medium. Comparison of the peak intensities gives the relative abundance of the protein(s) grown in the “heavy” and “light” medium.
Genetic tags
A large and growing class of tagging technologies uses genetic manipulation to attach a protein tag to your protein of interest. Such tags fall into three basic categories, but all work essentially the same way. First, clone your protein in-frame, either upstream or downstream of the desired protein tag. Then introduce the resulting plasmid into your expression cell line, and let the cells produce your tagged protein for you.
The first category is fluorescent protein tags. Autofluorescent proteins like green fluorescent protein (GFP) and its many variants (for instance, red, cyan, and yellow fluorescent proteins, not to mention the multichromatic palette devised by Roger Tsien at the University of California, San Diego) may be used to monitor protein expression and trafficking in vivo. Clontech offers several fluorescent protein cloning vectors, including a subset of Tsien’s palette (including mCherry and tdTomato), under its Living Colors® brand.
Vectors also exist for the coupling of purification tags to proteins. Such labels run the gamut from simple peptides (such as hexahistidine or the influenza hemagglutinin peptide tag) to full-length, intact proteins (like glutathione-S-transferase, GST), all of which can be used as purification handles, either via an antibody or affinity column.
According to Corina Nikoloff, product manager at Clontech Laboratories—which provides both His- and GST-tagging vectors—GST provides excellent purity and simplicity, but is also a somewhat troublesome tag.
“Some people love GST, but GST is huge—a 26 kDa whole protein,” she says. “Some proteins will not express with such a big tag, or will be exposed to degradation. The advantage of the GST tag is, when the protein does work, it’s very easy to purify with a glutathione column.” In contrast, the histidine tag—which enables affinity purification on nickel or cobalt resin—is tiny (either six histidine residues or 12 alternating histidines and asparagines), and can be purified to high abundance; the company recently released a resin whose binding capacity is 60 mg/ml, Nikoloff says.
The final class of genetic tags is what you might call “modifiable” protein tags. Available from New England Biolabs (SNAP-tag, CLIP-tag, ACP-tag, and MCP-tag), Life Technologies (Lumio), Promega (HaloTag), and Active Motif (LigandLink), these vectors each create a fusion of your protein of interest to a second protein that can then interact with a tagging reagent, either in vivo or in vitro, and either covalently or non-covalently. It’s like a molecular Swiss Army knife: from a single cloning event, you can generate multiple different tagged forms of the same protein.
“You can study the protein in a cellular and biochemical environment, with the same genetic construct,” says Paula Phenix, proteomics product manager at Promega.
Active Motif’s LigandLink™ Universal Protein Labeling system, for instance, couples your protein of interest to the E. coli DHFR (dihydrofolate reductase), which non-covalently binds the antibiotic trimethoprim. Researchers can label transfected cells when desired simply by adding trimethoprim-labeled fluorescent dyes; the dyes are cell permeable, so they can enter living cells, and both red and green variants are available. Similarly, Life Technologies’ Invitrogen-branded TC-FlAsH system uses a Cys-Cys-X-X-Cys-Cys hexapeptide to “coordinate” either of two diarsenic-containing fluorophores to a protein of interest.
The advantage of such a system, says Salvatore Russello, manager of global business development for New England Biolabs, is the on-demand flexibility it provides. “GFP is always fluorescent, so it’s on whether you want it on or not,” he says. “With our technology, you determine when you want the fusion protein to be fluorescent by adding the substrate.”
You can also determine the fluorescent color you desire (for instance, red or green) on a per-experiment basis, and use the same expression vector to facilitate fluorescent analysis and purification studies. Plus, you are no longer limited to the fluorescent properties of fluorescent proteins, but rather can expand your color palate with the organic dyes you already use in flow cytometry, for instance.
Promega’s HaloTag system, based on a 33 kDa monomeric bacterial hydrolase that covalently binds to a synthetic chloralkane ligand, includes modified reagents for both live and fixed cell fluorescent labeling, as well as such solid supports as Sepharose™, magnetic beads, and glass slides. NEB offers 14 cell-impermeable fluorophores for its SNAP-tag, as well as six cell-permeable dyes.
NEB’s four tags fall into two distinct classes, self-labeling and non-self-labeling. SNAP-tag and CLIP-tag are variants of a DNA repair enzyme that will covalently link your protein of interest to a fluorophore, bead, or capture reagent like biotin by addition of benzylguanine- or benzylcytosine-coupled reagents, respectively. ACP- and MCP-tagged proteins require the addition of a recombinant synthase enzyme to transfer the labeling reagent from derivatives of coenzyme A to the fusion tag.
The availability of these orthogonal tags, says Russello, gives users greater flexibility in experimental design, because the different tagging reagents (for instance, benzylguanine and benzylcytosine) don’t cross-react; thus, it becomes possible to label two different proteins in the same cell at the same time.
Chemical modification in vitro
The final class of tagging systems works in vitro, once a given protein has been extracted and purified. One classic application: applying a fluorescent tag to home-made antibodies for use in immunofluorescent studies.
A number of companies provide reagents for modification of proteins (especially antibodies) via NHS ester or maleimide chemistry; the former modifies the primary amines of lysine residues, while the latter attacks cysteine sulfur centers. Dojindo, for instance, offers kits that use NHS ester and maleimide chemistry to couple enzymes (for immunohistochemistry), biotin (for pull-down assays), fluorophores, or fluorescent proteins.
Life Technologies and Thermo Fisher Scientific offer reagents for in vitro modification of proteins for mass spectrometric analysis. Life Technologies’ Applied Biosystems-branded ICAT reagents, for instance, specifically label cysteine-containing proteins, facilitating two-sample multiplex analyses.
Purkayastha explains that ICAT was designed to specifically isolate cysteine-containing peptides, thereby reducing sample complexity. Thus, the tag contains both a cysteine-reactive group, as well as an affinity moiety for isolation of the tagged peptide.
Life Technologies’ Applied Biosystems-branded isobaric iTRAQ reagents, by contrast, is a global labeling strategy that uses NHS ester chemistry to label all proteolytic peptides (not just those containing cysteines) with tags that can be distinguished during tandem mass spec analysis, enabling up to 8-plex experiments. Thermo Fisher Scientific’s isobaric TMT reagents support up to 6-plex multiplexing.
For those interested in studying post-translationally modified proteins, reagents exist to “swap” specific modifications (such as palmitoylation or nitrosylation) with biotin, enabling protein fractionation, says Farooqui. Alternatively, you can feed cells azide-functionalized sugars to select glycoproteins via the Staudinger reaction.
As Jason Little, marketing and sales representative at Dojindo Molecular Technologies, points out, each of these reagents is really only necessary for those working on proteins for which pre-made reagents aren’t available. Still, he says, even if commercial reagents do exist, “our kits give a bit more flexibility.”