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.