Delivery of bioactive, gel-isolated proteins into live cells.
The delivery of proteins into live cells is a promising strategy for the targeted modulation of protein-protein interactions and the manipulation of specific cellular functions. Cellular delivery can be facilitated by complexing the protein of interest with carrier molecules. Recently, an amphipatic peptide was identified, Pep-1 (KETWWETWWTE WSQPKKKRKV), which crosses the plasma membrane of many cell types to carry and deliver proteins as large as antibodies. Pep-1 effectively delivers proteins in solution; but Pep-1 is not suitable for delivering sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolated proteins because Pep-1 complexes with cargo proteins are destroyed by SDS. Here, we report cellular delivery of SDS-PAGE-isolated proteins, without causing cellular damage, by using a nonionic detergent, Triton X-100, as carrier. To determine the specificity of our method, we separated antibodies against different intracellular targets by nonreducing SDS-PAGE. Following electrophoresis, the antibody bands were detected by zinc-imidazole reverse staining, excised, in-gel refolded with Triton X-100, and eluted in detergent-free phosphate-buffered saline. When overlaid on cultured NIH 3T3 cells, the antibodies penetrated the cells localizing to their corresponding intracellular targets. These results are proof-of-principle for the delivery of gel-isolated bioactive proteins into cultured cells and suggest new ways for experimental protein therapy and for studying protein-protein interactions using gel-isolated protein.
Department of Biochemistry, The University of Alberta, Edmonton, Alberta, Canada.
Jennifer E
JE
Taylor
Carlos
C
Fernandez-Patron
D000818:Animals; D000906:Antibodies; D002463:Cell Membrane Permeability; D002477:Cells / Q000378:metabolism*; D003584:Cytological Techniques / Q000379:methods*; D004337:Drug Carriers / Q000493:pharmacokinetics; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D041681:NIH 3T3 Cells; D017830:Octoxynol / Q000493:pharmacokinetics; D017510:Protein Folding; D011506:Proteins / Q000302:isolation & purification / Q000493:pharmacokinetics*
D000906:Antibodies; D004337:Drug Carriers; D011506:Proteins; D017830:Octoxynol