Abstract
Induced internalisation of functional proteins into cultured cells has become an important aspect in a rising number of in vitro and in vivo assays. The endo-lysosomal entrapment of the transduced proteins remains the major problem in all transduction protocols. In this study we compared the efficiency, cytotoxicity and protein targeting of different commercially available transduction reagents by transducing a well-studied fluorescently labelled protein (Atto488-bovine serum albumin) into cultured human sarcoma cells. The amount of internalised protein and toxicity differed between the different reagents, but the percentage of transduced cells was consistently high. Furthermore, in all protocols the signals of the transduced Atto488-BSA were predominantly punctual consistent with an endosomal localisation. To overcome the endosomal entrapment, the transduction protocols were combined with a photochemical internalisation (PCI) treatment. Using this combination revealed that an endosomal disruption is highly effective in cell penetrating peptide (CPP) mediated transduction, whereas lipid-mediated transductions lead to a lower signal spreading throughout the cytosol. No change in the signal distribution could be achieved in treatments using non-lipid polymers as a transduction reagent. Therefore, the combination of protein transduction protocols based on CPPs with the endosomolytic treatment PCI can facilitate protein transduction experiments in vitro.
Highlights
In cell culture experiments, the transduction of functional proteins into cells has become an alternative strategy with respect to transient transfection of the cells with specific gene expression vectors
The transduction efficiency is often measured as the total fluorescence intensity of cell lysates after transduction of a labelled protein measured by a fluorescence reader
These fluorescence intensities varied in the sarcoma cell lysates of Atto488-BSA treated cells depending on the used transduction reagents (Figure 1)
Summary
The transduction of functional proteins into cells has become an alternative strategy with respect to transient (or stable) transfection of the cells with specific gene expression vectors. Current protocols use different strategies to overcome the cellular membrane, essentially basing on cell penetrating peptides, lipid shuttle systems, non-lipid (cationic) polymers or endosomolytic reagents. Starting with the discovery of the protein transduction domain of the TAT protein from HIV, various short peptide sequences have been found to be able to enter living cells [1]. These so called cell penetrating peptides (CPPs) have been shown to be capable of transporting attached cargos into a wide range of cultured cell types [2]. Overcoming the endo-lysosomal entrapment remains the major problem in published transduction protocols of proteins
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