Six years ago, when John Tinsley's postdoctoral advisor at Texas A&Mtold him to find a protocol to introduce proteins into coronary endothelial cells, he couldn't find one in the literature. Tinsley tested a variety of commercial DNA transfection reagents, found one that worked for proteins, and has been using it since then.
Tinsley's experience would be a lot different today. No longer must researchers rely on tedious and toxic procedures, or on proprietary reagents designed specifically for DNA. Instead, the market is growing for so-called protein-transduction reagents, products specifically designed to introduce proteins and peptides into cells.
Though still a relatively small marketplace in comparison to nucleic acid delivery, the field has more than doubled since our last roundup.1 At that time the competition consisted solely of Active Motif and Invitrogen, both of Carlsbad, Calif., and Gene Therapy Systems and Imgenex, both of San Diego. Today's listing adds Avanti...
LIPIDS AND THE LIKE
Tinsley, now a research assistant professor, relies on TransIT-LT1, a plasmid-delivery reagent from Mirus of Madison, Wisc., to transfect peptides and large proteins. Others have used products such as Invitrogen's Lipofectamine. These and other formulations are marketed as ways of coating negatively charged DNA with polycations to make it less hydrophilic. The complex can then interact with the cell to be internalized, probably by membrane fusion, endocytosis, or both.
Neither Mirus nor Invitrogen markets or supports any of its DNA transfection reagents for protein transduction, company representatives say. Yet there are formulations specifically designed to get proteins across cell membranes. The first of these on the market was BioPorter from Gene Therapy Systems, says Mike Vengrow, GTS technical support manager. The cationic lipid formulation encapsulates the protein and binds to the cell surface, delivering a transfection efficiency of 70–90%, he says. Stratagene markets BioPorter as Biotrek; Imgenex markets it as ProVectin (outside the United States).
Pierce Biotechnology offers ProJect, a mixture of cationic lipids, to introduce proteins into cells, and MP Biomedicals offers TransPro. Targeting Systems offers two such products, Profect-1 and Profect-2. These are "neither lipid nor peptide," says Rampyari Walia, a principal at the company, who declined to disclose the class of the cationic compound. Similarly, ProteoJuice from EMD Biosciences is a "nonlipid" cationic reagent, also proprietary.
It's the mysterious nature of all these reagents that frustrates Paul Wender, a drug-delivery researcher at Stanford. "One of the problems with working with a commercial system is that it either works or it doesn't work," he says. "If it doesn't work, since we don't know what the proprietary mixture is, we often can't fix the problem."
This same reasoning stopped phosphatidyl and sphingolipid manufacturer Avanti Polar Lipids from putting together a protein transduction kit, despite requests. Customer feedback about other kits had convinced Shaw that too many proteins and too many cell types preclude a one-size-fits-all solution to protein delivery.
PROTEIN TRANSDUCTION DOMAINS
Some researchers ferry cargo across the plasma membrane not with cationic reagents but with short peptides, called protein transduction domains (PTDs). Highly basic sequences in the HIV tat protein, and similar PTDs in a variety of organisms, allow proteins harboring these tags to be rapidly taken up by cells in a receptor- and transporter-independent fashion. PTDs probably form an ionic interaction with the plasma membrane, followed by a multistep process resulting in rapid internalization by macropinocytosis, destabilization of the macropinosome, and release into the cytoplasm.2
Qbiogene uses a 16-amino acid peptide derived from
Invitrogen offers two ways to use its Voyager system, which is based on the herpes simplex virus structural protein VP22: Clone a gene for the protein of interest into either a bacterial expression cassette or a eukaryotic expression vector. The fusion protein can be purified directly from cell lysates and added to the cell culture. Or, transfected cells can be used to produce fusion protein directly in the culture itself, where it will spread to surrounding cells. Transfected fusion proteins will accumulate in the nuclei of transfected cells, unless the vector encodes a nuclear export signal.
Active Motif's Chariot reagent works via a different mechanism than do other PTDs: The 25-amino acid peptide noncovalently coats the protein to be transfected, "like a dandelion seed," says Eric Simon of Active Motif. The peptide "hairs" invade the cell membrane and ferry in their cargo. Once inside, the complex dissociates, leaving a native, intact protein.
POKE IT, ZAP IT, TARGET IT
Microinjection and electroporation are still used at times to deliver proteins to cells, although both methods have severe limitations. The former requires considerable skill and is very labor intensive. As for the latter, about half the cells subjected to an electric shock sufficient to introduce a macromolecule die from the treatment.
Courtesy of Active Motif
Active Motif's Chariot reagent is one of a growing pool of formulations available to deliver proteins to cells. The protocol is simple enough: Mix protein with the reagent, incubate, add to cells, wait a little while, and finally assay. The inset box provides an example, showing human fibroblast (HS-68) cells transfected with an anti-actin antibody. These unfixed cells were observed one hour later.
In a new twist to an old concept, IPBio Sciences recently introduced its Immunoporation system. Magnetic beads coupled to antibodies to a cell surface antigen are mixed with cells and protein. When the mixture is subjected to a magnetic field, small holes transiently form in the cell membrane, allowing entry of the protein. "The critical advantage" to this technology, says CEO Tom Hole, "is the ability to hit a particular cell type." Only cells with the targeted antigen will be disrupted.
Yet creating holes in cells, however transient, is not without its problems. "The reason why we and others are a little less bullish on those kinds of things," notes Wender, "is that as soon as you start changing the integrity of the membrane, you allow not only what you want to get in, but you allow other things to get in, and you allow many things to get out." He notes as an example that "poking holes in cells" can result in ion leakage and a change in membrane potential.
Researchers are currently looking into finding new ways to facilitate protein entry without compromising the barrier. Some make use of natural cell-surface structures that are known to internalize with their cargo upon binding, such as the folate receptor, a chemotherapy target. Blake Peterson of Pennsylvania State University has taken that idea one step further by synthesizing cholesterol-amine-based receptors designed to specifically recognize the protein to be transfected. These receptors integrate into the membrane, facilitating uptake by the cell.
LIMITATIONS AND CONSIDERATIONS
Though the protein transduction field has grown substantially since Tinsley's post-doc days, there remain many issues that users must consider. Researchers must make sure their cargo reaches its intended destination intact, which can often be done by means of a reporter molecule such as a fluorescent dye or protein. Endosomes, formed when cargo is internalized, for example, can fuse with lysosomes. Membrane fusion could, perhaps, leave the cargo sitting on the outside of the cell. Some PTDs are linked to a nuclear localization sequence, requiring an additional nuclear export signal to deliver cargo to the cytoplasm.
Cargo size, and perhaps charge, need to be compatible with the reagent. Though several of the companies offering protein-transduction tools claim their reagents are virtually limitless in this regard, others restrict their claims to oligopeptides.
Fusing the protein to a PTD, or exposing it to denaturing agents, may add an additional layer of complexity. Similarly, agents that compromise the integrity of the cell (including polycations) may complicate data analysis. Finally, logistics such as the amount of time required to set up and perform a reaction, and the transduction efficiency, should be taken into consideration.