teraction was shown in the nucleus for the first time, offers additional support that the protein was trafficked correctly by the host.
Secreted effectors from pathogenic bacteria have evolved to function within the host cell environment and thus it is helpful to study them in situ within the host. Introduction of specific effectors of interest into host cells allows the relevant pathogen-host interactions to be studied in isolation without interference from other bacterial proteins. The goal was to explore electroporation as a means to introduce bacterial effector proteins into eukaryotic host cells, thereby avoiding some of the challenges associated with transfection or transduction. Green fluorescent protein was used as a control and Salmonella effector proteins were tested. Because of interest in bacterial pathogens that target host epithelial cells as well as immune cells, a human epithelial-like cell line (HeLa) and mouse macrophage-like cells (RAW 264.7) were used. Electroporation can deliver exogenous proteins into host cells with no appreciable decrease in cell viability, and the delivered proteins were detectable in cells for up to four days.
To isolate the effects of electroporated protein, pure protein is required. In this study, proteins were expressed in E. coli strain BL21 with N-terminal polyhistidine and streptavidin binding-peptide tags. Proteins were purified with Ni-NTA resin, assayed for concentration (generally between 5-25 mg/ml) and purity, and stored at -80 °C until electroporation. Commercially produced proteins would also be acceptable for electroporation, as they tend to be of high purity and well characterized.
Key steps in this protocol included completely removing the cell culture medium by washing and suspending cells in protein-free buffer. This ensures that any downstream phenotypes observed are due to the exogenous protein of interest and not to proteins present in the culture medium. Better efficiency of electroporation was observed when cell density was higher (e.g. 6 x 106 vs. 1 x 106 cells), though the best cell number will vary with cell size and type. Another critical step in the experiment was to allow cells time to recover and reattach to dishes; this was necessary because adherent cells were used in this study. A recovery period should be less important for suspension cells, although it may be necessary to give proteins time for proper intracellular trafficking, protein-protein interactions, or enzymatic activity, depending on the nature of the intended study. Since delivered proteins were observed inside cells for up to 96 hr, there is much room for adjustment to the incubation period prior to microscopy visualization or cellular assay.
Several variables in this protocol may be optimized for different cell types, protein targets, or downstream analysis schemes. The amount of protein to be electroporated can be fine-tuned for the desired intracellular concentration. For visualization of protein localization, as little as 1 µg can be used. For functional studies, higher starting amounts of proteins may be needed. In addition, the amount of time for post-electroporation cell recovery can be shorted or extended. Labile protein targets or fast downstream processes would require a shorter incubation time. Also, the amount of cells plated can be adjusted beyond the suggestions here. Cells may be plated more sparsely if the introduced protein is to be monitored by microscopy, or plated more densely if the electroporated protein is to be recovered for applications such as western blots.
While electroporation is a simple and straightforward method for introducing proteins into living cells, there are some limitations to the technique. The method requires highly pure proteins in microgram amounts. As little as 1 µg was electroporated and visualized with confocal microscopy, but higher starting amounts of protein gave better visual results. While protein function was not assessed at all concentrations after electroporation, protein concentrations equal to or greater than 50 μg/ml were req