Abstract
Direct delivery of recombinant proteins into mammalian cells is an experimental approach that shows great potential for protein localization and function studies. Microinjection, which is currently the widely-used method for introducing recombinant proteins into mammalian cells, requires considerable handling skills and is a low-throughput approach limiting the delivery to a small number of targeted cells. On the other hand, high throughput delivery methods, like cell-penetrating peptide tags or pore-forming bacterial toxins, display uncertain protein uptake efficiencies and varying degrees of cytotoxicity. Here, we describe a protocol for batch electroporation (EP) which is suitable for the delivery of single polypeptides and multi-subunit protein complexes into mammalian cells. To perform EP, cells are harvested, washed and re-suspended in an electroporation buffer containing the diluted protein (called the “EP slurry”). The EP slurry is subjected to multiple electric pulses that create reversible ruptures in the cell’s membrane and allow intracellular protein delivery. In our detailed study of the applications of the method, we show that EP displays homogenous uptake efficiency, semi-quantitative delivery of protein and rather low levels of cytotoxicity over multiple cell lines.
Keywords: Protein delivery, Live imaging, Fluorescent proteins, Cell biology, Biochemical reconstitution, Electroporation
Background
Traditionally, microinjection is the most commonly used method for the introduction of recombinant proteins into mammalian cells (Komarova et al., 2007). This approach, even though it serves the purpose, has major limitations such as its low-throughput and the requirement of considerable handling skills. High-throughput approaches that enable protein delivery to a large number of cells are available, for instance the use of cell-penetrating peptide (CPP) tags (Inomata et al., 2009) or pore-forming bacterial toxins (Ogino et al., 2009; Teng et al., 2016), but present uncertainties with regard to protein uptake efficiencies and cytotoxicity. We extensively studied Batch Electroporation (EP) as a method for direct delivery of recombinant proteins into cultured human cells. The protocol that we describe does not demand advanced handling skills and it is easily and rapidly implemented. EP works efficiently on diverse mammalian cells, provides low cytotoxicity levels, homogenous uptake efficiencies between individual cells and it is semi-quantitative. We successfully employed EP as a delivery method for a number of cell biology applications (Alex et al., 2019). We observed that EP delivered recombinant proteins achieved native localization, physically interacted with endogenous binding partners and functionally complemented RNAi depleted endogenous complexes. EP is also suitable for live-cell spectroscopic applications such as FLIM-FRET microscopy or FRAP analysis. Moreover, EP can be used to introduce proteins with modified chemical handles or functional probes into cells and identify the elusive binding partners. Thus, in addition to its ease of application, EP is very attractive because it allows delivery in a sufficiently large cohort of cells and is therefore an ideal tool to bridge in vitro reconstitution and cell biology studies.
Materials and Reagents
Equipment
Procedure
Electroporation (EP):
Notes
Recipes
Acknowledgments
We thank Melina Schuh and Dean Clift for discussion and comments. We are grateful to all members of the Selenko and Musacchio laboratories for reagents, helpful discussions and comments. A.M. gratefully acknowledges funding by the Max Planck Society and the European Research Council (ERC) Advanced Investigator Grant RECEPIANCE (proposal number: 669686).
Competing interests
All authors have no conflict of interest to declare.
References
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