New type Protein Transfection Reagent ProteoCarry | Funakoshi

Going to Cytoplasm, not ending up in endosome!
ProteoCarry

ProteoCarry is a new type peptide base transfection reagent.

When using a conventional protein transfection reagent, transfected proteins are accumulated in endosome or lysosome, and most of such proteins are dissociated in lysosome proteolysis system.
Contrary, ProteoCarry can deliver transfected proteins to cytoplasm effectively.
Moreover, pre-incubation with proteins are NOT needed.
Therefore effect to the protein functions can be minimized.

Transfection of fluorophore-conjugated dextran
HeLa cells were treated with green fluorophore-conjugated dextran (Dextran-FL, average MW 10 kDa, final 200 μg/ml) in the presence or absence of ProteoCarry (1x conc.) for 1 hour at 37oC. Without ProteoCarry, fluorescent signals were detected from endosome-like dot structures inside cells. On the other hand, ProteoCarry clearly stimulated cytosolic delivery of Dextran-FL.

Transfection of fluorophore-conjugated IgG
HeLa cells were treated with green fluorophore-conjugated IgG (IgG-FL, final 50 or 500 μg/ml) in the presence or absence of ProteoCarry (1x conc.) for 1 hour at 37oC. Without ProteoCarry, IgG-FLs were aggregated into endosome-like dot structures. On the other hand, ProteoCarry promoted cytosolic delivery of IgG-FL even if some IgG-FLs were detected in endosomes.

Transfection of fluorophore-conjugated anti-nucleus pore complex (NPC)
HeLa cells were treated with red fluorophore-conjugated anti-nucleus pore complex (NPC) antibody (anti-NPC-FL, final 50 μg/ml) in the presence or absence of ProteoCarry (1x conc.) for 1 hour at 37oC. Without ProteoCarry, a little of fluorescent signal was observed in dot-like structure. On the other hand, ProteoCarry clearly promoted localization of anti-NPC-FL to peri-nuclear structure. This result indicates that ProteoCarry could induces incorporation of anti-NPC-FL into cytosol and binding of antibody to nucleus pore complex.

Transfection of nuclear-localizable EGFP
HeLa cells were treated with nuclear localization signal (NLS)-tagged EGFP (NLS-EGFP, final 10 μM) in the presence or absence of ProteoCarry (1x conc.) for 1 hour at 37oC. Without ProteoCarry, a little of fluorescent signal was observed in endosome-like dot structures. On the other hand, ProteoCarry promoted nuclear-localization of NLS-EGFP.

Transfection of Cre recombinase for Cre/lopX recombination assay
HeLa cells were transfected with a plasmid which encodes loxP-DsRed-stop-loxP-EGFP sequence by conventional lipofection reagent. Next day, the HeLa cells were treated with recombinant Cre recombinase (final 10 μM) for 1 hour at 37oC with or without ProteoCarry (1x conc.). Without ProteoCarry, almost cells still keep DsRed expression under the treatment of Cre recombinase. On the other hand, ProteoCarry promoted conversion from DsRed to EGFP by Cre/loxP recombination.

Transfection of Saporin enzyme
Saporin is an enzymatically cytotoxic protein which binds to ribosomes and inactivates ribosomes via its RNA glycosidase activity. RAW264.7 cells were treated with saporin (final 10 μg/ml) with or without ProteoCarry for 1 hour at 37oC. After 24 hours culture, cell viability was measured by MTT assay. Without ProteoCarry, saporin induced cell death with less than 30% efficiency. On the other hand, ProteoCarry dramatically increased cytotoxicity of saporin and almost cells were dead (>90%).

Reference data: Intercellular localization of FL-labeled ProteoCarry
To monitor intracellular localization of ProteoCarry, fluorophore-labeled ProteoCarry (ProteoCarry-FL) was prepared and HeLa cells were treated with ProteoCarry-FL. Fluorescent signal was observed by confocal microscopy. Signal was detected mainly in endosomal dot like structure and little fluorescent signal was observed in cytosol. This data indicates ProteoCarry keeps binding to endosomal membrane after lysis of endosomes.