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Feature Articles : Jan 1, 2008 ( )
Tech Note: Delivery of Functional Proteins Inside Cells
Two New Reagents Deliver Proteins into Cells Without Sensitivity to Serum
Cell transfection with nucleic acids is the best way to express or to inhibit a protein inside cells. Efficient transfection of many primary cells and cell lines is possible as the result of considerable advances in technologies and reagents over the past few years.
Experimental approaches to regulate protein function or expression usually target gene function at the DNA and/or mRNA level. These methods are powerful but not perfect. If a protein has several different functions, for example, it is difficult or even impossible to selectively inhibit only one of them. Membrane-permeable drugs can be used to regulate a specific protein function but their lack of specificity and off-target effects are an issue.
For these reasons, delivery systems that allow exogenous native or mutant proteins to be transported inside living cells represent a workable solution for functional studies or new therapeutic assays. Such a system could open new fields of investigation in proteomics and also more accurately elucidate molecular mechanisms.
The delivery of antibodies into living cells appears to be a promising way to study intracellular trafficking as a result of various stimuli or to specifically block only one function of a protein and/or protein-protein interaction. Inefficient folding and assembly in the cytosol currently limits antibody use in living cells, however, the use of antibodies from the Camelidae species has been proposed as a solution to this problem. Camelidae antibodies are devoid of light chains, which makes folding in the cytosol possible.
While the physico-chemical properties of nucleic acids are identical, the hydrophobicity pattern and charge distribution of proteins varies considerably and constitutes a significant challenge for protein interactions with cationic lipids.
The protein to be delivered has to cross the cellular membrane to make it into the cytoplasm, which constitutes another challenge. Strategies to overcome this hurdle include the use of membrane-permeable peptide carriers such as PTD (protein transduction domain), which have been shown to transduce cargo across the plasma membrane.
These PTDs, unfortunately, interact poorly with proteins, and covalent chemical or genetic linkage between the PTD and the cargo is usually required. Nevertheless, the use of a long synthetic peptide issued from two domains of the Tat and GP120 proteins of HIV-1 was shown to interact noncovalently with some proteins and transduce them across the plasma membrane.
Several liposomal formulations have been developed to transfer proteins through electrostatic and hydrophobic interactions and deliver them inside cells. Their efficiency is still limited though, and they require laborious optimizations, which limit widespread use.
Protein Delivery in Living Cells
Oz Biosciences (www.ozbiosciences.com) has developed a lipid-based formulation designed to interact with proteins and introduce them inside the cytoplasm of living cells. Many molecules and formulations were tested; some were able to interact efficiently with the assayed proteins, but few were able to deliver them inside cells. SM181 allowed the efficient delivery of several proteins in different cell lines. In fact, proteins rapidly accumulated inside the cytosol in four to five hours and appear as a diffuse label (Figure 1A, B, C, D). The use of proteins without SM181 did not lead to any fluorescent signal inside the cells.
Surprisingly, the presence of serum does not interfere with the protein delivery assay but helps to obtain more reproducible results instead. It appears that complexes between proteins and SM181 are smaller when serum is present during the incubation with cells, which could facilitate uptake. Down the road this feature could allow in vivo applications, which is not foreseeable with existing reagents.
Additionally, the delivered protein is still functional since the enzymatic activity of the b-galactosidase is maintained upon delivery (Figure 1A). This was confirmed by the delivery of an active caspase-3 inside cells.
The goal of this experiment was to study the impact of delivery of an apoptosis-inducing protein on cell physiology. After six hours of incubation, most of the cells were detached from the culture dish or showed a nonphysiological round shape (Figure 1E and F). The annexin-V-FITC staining confirms that almost 50% of the cells were apoptotic (Figure 1G).
The formulation and composition of SM181 was further refined to improve antibody delivery. The resulting new lipid formulation (Ab-SM181) allows delivery of FITC-labeled antibodies in the cytosol (Figure 2A) with high efficiency. The vast majority of the cells were positive after only a few hours of incubation, and optimum delivery was reached at four to six hours (Figure 2D).
Two different specific antibodies were used in additional research to see if they were able to reach and recognize their target inside the cell. The first one was directed against the cytosolic domain of giantin, a large transmembrane protein localized to the Golgi apparatus. The second one was directed against some proteins of the nuclear pore complex (NPC). The antigiantin antibody localized to an area close to the nucleus as a punctuate stain (Figure 2B), showing it is functional and able to reach its intracellular target. This result has been confirmed by the use of the anti-NPC antibody, which localized to and stained the nuclear envelope (Figure 2C).
After two to three hours of incubation, both antibodies showed diffuse staining in the cytosol before they accumulated in the expected area. This accumulation is not only time dependent but also cell-type and protein-concentration dependent. Consequently, it will take a longer time to observe a specific staining if high amount of antibodies are delivered.
In another experiment, we verified that NLS-bearing antibodies were able to localize and accumulate inside the nucleus confirming that the proteins are freely released and fully functional inside cells (data not shown). The protocol is a fast three-step procedure.
The protein is directly added onto the SM181 formulation in a microtube under sterile conditions. The mixture is then incubated for 10–15 minutes at room temperature before being incubated with the cells growing in their regular culture medium.
SM181, unlike all the existing protein delivery reagents, did not appear sensitive to the presence of serum during this last step and thus did not require incubation in serum-free medium. Instead, the presence of serum during incubation appeared to reduce the aggregation of complexes and favored protein uptake inside cells.
SM181 is able to interact by electrostatic and/or hydrophobic interactions with proteins to form macromolecular complexes. In addition, it efficiently promotes the delivery of proteins to a wide variety of cells including primary cells. This reagent is not toxic and because it can be used in the presence of serum it represents an important improvement over existing protein delivery reagents.
One proposed application—delivery of blocking antibodies inside living cells—could facilitate completion of studies realized with siRNA or mutated proteins. Such an approach could also prove to be a good alternative when cells are not efficiently transfected with DNA, such as primary neurons. In our studies, we were able to deliver proteins in more than 70% of primary neurons and glial cells, whereas DNA transfection efficiency is much lower.
Laurent Meunier, Ph.D., is R&D cell biology department manager, Stephane Moutard, Ph.D., is the manager of the chemistry department, and Olivier Zelphati, Ph.D., is the CEO of Oz Biosciences. Web: www.ozbiosciences.com. E-mail: firstname.lastname@example.org.
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