Genetic engineering of proteins with cell membrane permeability.
The discovery of methods for generating proteins with inherent cell membrane-translocating activity will expand our ability to study and manipulate various intracellular processes in living systems. We report a method to engineer proteins with cell-membrane permeability. After a 12-amino acid residue membrane-translocating sequence (MTS) was fused to the C-terminus of glutathione S-transferase (GST), the resultant GST-MTS fusion proteins were efficiently imported into NIH 3T3 fibroblasts and other cells. To explore the applicability of this nondestructive import method to the study of intracellular processes, a 41-kDa GST-Grb2SH2-MTS fusion protein containing the Grb2 SH2 domain was tested for its effect on the epidermal growth factor (EGF)-stimulated signaling pathway. This fusion protein entered cells, formed a complex with phosphorylated EGF receptor (EGFR), and inhibited EGF-induced EGFR-Grb2 association and mitogen-activated protein kinase activation.
Rojas
M
M
Department of Microbiology and Immunology, Vanderbilt University, Nashville, TN 37232, USA.
Donahue
J P
JP
Tan
Z
Z
Lin
Y Z
YZ
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D002463:Cell Membrane Permeability; D006224:Cricetinae; D005982:Glutathione Transferase / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D011506:Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase