Getting Proteins Into Cells

A postdoctoral fellow has just identified an interesting new gene. But to get published in a top-flight journal, there's a need to figure out what the gene product does in vivo. Unfortunately, to accomplish that, the postdoc needs a way to get the protein into the cell, and therein lies the problem: There are many fast and effective methods to introduce transcriptionally active DNA into cells,1 but options for delivering functional proteins into cells are limited. New research and commercially available products are offering scientists another approach: protein transduction. Protein transduction is the process by which peptide or protein motifs cross the cellular plasma membrane.2 The direct application of functional peptides and proteins to cells has been used to probe signal transduction pathways, block transcription factors, and conduct detailed structure/function analyses of integrin and other receptors' cytoplasmic domains, among other research applications.3 One of the main advantages of protein transduction over DNA transfection as a method to express proteins in cells is that, whereas some mammalian cells are notoriously difficult to transfect, all mammalian cells tested to date are receptive to protein transduction. In addition, transduction occurs rapidly, with the protein detected in about 10 minutes rather than after several hours or days. Finally, by varying the amount of protein added to the culture medium, researchers can control the final intracellular protein concentration. Protein transduction is not the only protein-delivery method. Scientists can achieve the same effect using other, more invasive techniques. But the applicability of protein transduction to a wide range of cell types, the speed and ease of translocation across the plasma membrane, and the lack of toxicity of the process, make transduction an attractive option. As a result of the recent explosion in transduction research, a growing range of protein delivery products and kits are coming onto the market. The Old-Fashioned Way The mammalian cell membrane is refractory to most proteins and peptides. Until recently, the most widely used methods to introduce antibodies, peptides, or other membrane-impermeable molecules into cells were microinjection and electroporation—both invasive techniques that disrupt the cell membrane. Microinjection involves loading or transferring a dissolved substance into a living cell using the microscopic tip of a glass capillary. The technique requires technical skill and can only be performed one cell at a time. Electroporation uses high-voltage pulses to produce transient pores through which proteins can enter. While this technique can be applied to many cells at once, it is toxic and non-specific—that is, anything can enter or exit the cell once the membrane is disrupted. Other limitations of these methods include the efficiency and cell-to-cell variability. Electroporation, for instance, delivers proteins to only 1-2% of a primary cell population and 10-15% of an established cell line population. Thus, the cells that do not receive the protein can easily mask any effect produced by those cells that do. Further, not all of the cells that take up protein end up with the same concentration of exogenous protein, leading to variable and difficult-to-interpret results. The serendipitous observation that some proteins can enter cells when added to the surrounding media, led to the identification of short basic peptide sequences from these proteins that can traverse the plasma membrane and carry the rest of the protein with them.4 The three most widely studied protein transduction domains (PTDs) are derived from the Drosophila homeotic transcription factor Antennapedia (Antp), the herpes simplex virus (HSV) protein VP22, and the human immunodeficiency virus (HIV)-1 transcriptional activator Tat. Other proteins, such as the Drosophila homeotic factors Fushi-tarazu and Engrailed, contain peptides with similar properties.5 Protein Transduction Domains PTDs are generally short peptides, about 10-16 residues in length. Structurally dissimilar, their only common feature appears to be the presence of numerous positively charged lysine and arginine residues.4 PTD-mediated transduction evidently occurs by a mechanism other than classical endocytosis: Cells can take up these peptides at 4°C and in the presence of cellular transport process inhibitors.4 PTD-mediated transduction seems to be independent of protein size. PTDs have delivered covalently attached proteins in excess of 700 kDa to cells. They have also transduced liposomes over 200 nm in diameter—about the size of a mitochondrion—directly across the cell membrane by anchoring PTDs to the liposome surface.6 If desired, the covalent attachments that link the PTD to its cargo can be made reversible, for example, by using disulfide or ester linkages that are reduced or cleaved once inside the cell. PTD-mediated Tat transduction occurs through a poorly defined mechanism that is independent of receptors, transporters, and endocytosis. When added exogenously to cells, the 101-residue-long Tat protein crosses the plasma membrane, localizes to the nucleus, and initiates the replication of viral DNA. Tat's PTD consists of an arginine-rich stretch of nine amino acids (RKKRRQRRR) embedded within the protein. When synthesized as a recombinant fusion protein or covalently cross-linked to other peptides or proteins, Tat is capable—albeit with poor efficiency—of delivering biologically active cargo to a cell. However, if the cargo is instead covalently linked at the N- or C-terminus to a truncated Tat PTD, the transduction efficiency improves dramatically. Transduction then occurs via a rapid process that targets cells in a concentration-dependent fashion at both 37°C and 4°C. Tat-coupled proteins have been transduced into cells and tissues in vivo, as well as in culture. University of California at San Diego associate professor Steven F. Dowdy, then at the Washington University School of Medicine in St. Louis, and his postdoctoral fellow Steven R. Schwarze demonstrated by intraperitoneal injection into a live mouse that the Tat peptide can deliver functional protein—in this case, b-galactosidase (120 kDa)—to all murine tissues tested, including the brain.7 Only proteins that are covalently linked to the PTD can get in and out of cells, thus ensuring that the process is specific—a particularly important consideration for in vivo applications of this technology. Structural analyses of naturally occurring PTDs have allowed researchers to design an estimated 50 to 70 synthetic, model PTDs, some as short as six amino acids.2 Dowdy and his group were among the first to demonstrate the delivery of synthetic PTDs to cells in culture, as well as in vivo. For example, his team developed a Tat derivative (YARAAARQARA) that is 33-times more efficient than the Tat peptide itself, and which can deliver small molecules in vivo.8 The HSV transcription factor, VP22, also contains a PTD. Carlsbad, Calif.-based Invitrogen Corp. (www.invitrogen.com) sells a number of vectors based on this protein, which it licensed from Cambridge, UK-based Phogen Ltd. (www.xenova.co.uk/phogen.html). VP22 can enter a range of mammalian cell types following expression and purification from Escherichia coli, and can migrate from cell to cell in a population once it does so. Invitrogen offers four protein production kits that researchers can use to express VP22 fusion proteins—with VP22 either at the protein's N- or C-terminus—in E. coli. In the absence of any localization signal on the protein, VP22 fusion proteins accumulate in the nucleus. However, two of Invitrogen's vectors have a nuclear export signal (NES) that enhances localization of VP22 fusions to the cytoplasm. A Non-Covalent Carrier Courtesy of Active Motif IncIntracellular Staining, Sans Fixing: Transduction of a FITC-labeled anti-actin filament antibody into cells with Active Motif's Chariot reagent. The image was taken 2 hours post- delivery and the cells were unfixed. Natural and synthetic PTDs must be covalently coupled to their cargo, which is a potential limitation of the technology. However, the Chariot™ reagent from Carlsbad, Calif.-based Active Motif Inc. (www.activemotif.com),9 is based on a different protein delivery system, which uses a short synthetic signaling peptide, called Pep-1, but which does not require covalent coupling to the protein or peptide cargo. Pep-1 is a 21-residue-long carrier consisting of three domains: a hydrophobic, tryptophan-rich motif that targets the cell membrane and forms hydrophobic interactions with proteins; a hydrophilic, lysine-rich domain derived from the simian virus 40 large T antigen nuclear localization sequence, which improves intracellular delivery and solubility of the peptide vector; and a spacer sequence.10 Courtesy of Gilles DivitaA Metaphase Spindle decorated with anti-tubulin (green) antibodies; chromsomes are stained with Hoechst (blue). The antibodies were delivered using Pep-1. When Pep-1 is mixed with peptides or proteins for 30 minutes in buffer, the molecules rapidly associate through noncovalent hydrophobic interactions and form stable complexes, each having an excess of carrier peptide over the cargo. For example, one molecule of green fluorescent protein (GFP; 30 kDa) interacts with 12-14 molecules of Pep-1.10 Scientists add these complexes to cells in culture, and two hours later, they can detect the protein in situ. Once inside the cell, Pep-1 and the cargo rapidly dissociate by a process called "decaging." The protein cargo is then free to move to its proper intracellular location. According to Gilles Divita, at the Centre de Recherches de Biochimie Macromoleculaire in Montpellier, France, whose group developed Pep-1, decaging takes about 30 minutes for small molecules and up to three hours for larger proteins. Courtesy of Active Motif IncHS-68 CELLS transduced with a Lucifer yellow-tagged, 10-kDa nuclear protein using Active Motif's Chariot reagent. This image was taken 90 minutes post-delivery, and shows unfixed cells. So far Divita's group has been able to transduce proteins up to 500 kDa, but other groups have transduced even larger molecules, including protein-DNA complexes. The ability of Pep-1 to deliver proteins in vivo is yet to be demonstrated and is, according to Divita, a focus of current research. Active Motif's Chariot reagent delivers proteins into mammalian cells at an average efficiency of 60-95%, depending on the cell type and the protein being transduced. The company is now testing Chariot for DNA or RNA delivery. Active Motif is also developing a peptide for the delivery of very small drugs, which would have a slow decaging time. Lipid Carriers While membrane-traversing peptide research is an area of intense activity, and the technology has shown great promise as a tool for protein delivery, other methods for noninvasive protein delivery are also being investigated. One avenue currently being explored is the use of lipid-based carriers. Liposomal carriers have been successfully used to deliver DNA into cells, but for the most part they have proven inefficient at functional protein delivery, transducing fewer than 5% of the population. Olivier Zelphati and colleagues at San Diego-based Gene Therapy Systems Inc. (www.genetherapysystems.com), however, have developed a new lipid formulation that interacts rapidly and noncovalently with protein, creating a protective vehicle for delivery.11 The protein encapsulated in the formulation binds to the negatively charged membrane, is internalized in endosomal vesicles by endocytosis, and is then released inside the cell. The product, sold as BioPORTER® by Gene Therapy Systems and as ProVectin™ by San Diego-based Imgenex (www.imgenex.com), consists of a dry film of reagent in a test tube to which scientists add the desired protein solution. After a five-minute incubation, serum-free medium is added, and the complete mixture is applied to cells for protein delivery. The transduction efficiency varies depending on the input protein and cell type used, ranging from 35-99%, with up to 50% of input protein introduced in the cells. The system displays no significant toxicity under optimal conditions. Its main limitation is that highly positively charged molecules with few or no hydrophobic domains—or containing inaccessible hydrophobic domains—will not be carried across plasma membranes. In addition, the system does not work in the presence of serum. Courtesy of Gene Therapy Systems Inc.BioPORTER Explained: Two separate mechanisms are shown. The BioPORTER-protein complex attaches to the negatively charged plasma membrane. It can then either fuse with the membrane to immediately deliver its cargo (1) or the complex can be endocytosed into an endosome, releasing the protein into the cell once the transduction complex fuses with the endosomal membrane (2). Therapeutic Applications One of the attractive features of protein transduction technology is its potential application to clinical medicine. While research in this field is still in its infancy, it is possible that protein delivery will have some application to current gene therapy protocols, in cases where the direct delivery of the gene product itself may be more beneficial then the delivery of the gene. In addition, protein transduction technology has potential applications for vaccine development. For instance, Phogen has licensed its VP22 technology to Palo Alto, Calif.-based Genencor International Inc. for the development of therapeutic vaccines against hepatitis B, hepatitis C, and human papilloma virus. Finally, the technology has potential drug delivery applications. Jonathan B. Rothbard and colleagues at Cellgate Inc. of Sunnyvale, Calif., have developed a range of synthetic peptide derivatives, or peptoids, that can be covalently linked to small drugs for their delivery and uptake by cells at different sites in the body.12 According to Rothbard these peptoids are more cost-effective, easier to synthesize, and more resistant to proteases than naturally occurring or synthetic peptides—features that make them more suitable to clinical applications. Cellgate is currently conducting the first human clinical trials of its products. Laura Bonetta (laura.bonetta@verizon.net) is a freelance writer in Garrett Park, Md. References 1. D.L. Stull, "New tools enable gene delivery," The Scientist, 14[24]:30, Dec. 11, 2000. 2. J.S. Wadia, S.F. Dowdy, "Protein transduction technology," Current Opinion in Biotechnology, 13[1]:52-6, Feb. 1, 2002. 3. J. Hawiger, "Noninvasive intracellular delivery of functional peptides and proteins," Current Opinion in Chemical Biology, 3:89-94, 1999. 4. S.R. Schwarze, S.F. Dowdy, "In vivo protein transduction: Intracellular delivery of biologically active proteins, compounds and DNA," Trends in Pharmacological Sciences, 21:45-8, February 2000. 5. K. Han et al., "Efficient intracellular delivery of GFP by homeodomains of Drosophila Fushi-tarazu and Engrailed proteins," Molecules and Cells,10:728-32, 2000. 6. V.P. Torchilin, "TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors," Proceedings of the National Academy of Sciences (PNAS), 98:8786-91, July 17, 2001. 7. S.R. Schwarze et al., "In vivo protein transduction: Delivery of biologically active protein into the mouse," Science, 285:1569-72, 1999. 8. A. Ho et al., "Synthetic protein transduction domains: Enhanced transduction potential in vitro and in vivo," Cancer Research, 61:474-7, Jan. 15, 2001. 9. M.A. Wingard, "Special delivery," The Scientist, 14[22]:19, Nov. 13, 2000. 10. M.C. Morris et al., "A peptide carrier for the delivery of biologically active proteins into mammalian cells," Nature Biotechnology, 19:1173-6, December 2001. 11. O. Zelphati et al., "Intracellular delivery of proteins with a new lipid-mediated delivery system," Journal of Biological Chemistry, 276:35103-10, Sept. 14, 2001. 12. P.A. Wender et al., "The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters," PNAS, 97:13003-8, 2000.

Getting Proteins Into Cells

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