Comparison of protein transduction domains in mediating cell delivery of a secreted CRE protein.
Protein transduction domains (PTDs) are versatile peptide sequences that facilitate cell delivery of several cargo molecules including proteins. PTDs usually consist of short stretches of basic amino acids that can cross the plasma membrane and gain entry into cells. Traditionally, to assess PTD mediated protein delivery, PTD-fusion proteins have been used as purified proteins. To overcome the requirement for a protein purification step, we used a secretory signal peptide to allow PTD-CRE fusion proteins to be exported from transfected mammalian cells. PTD induced protein transduction into cells was assessed by a CRE-mediated recombination event that resulted in beta-galactosidase expression. Several PTDs were tested including the prototypic TAT, different TAT variants, Antp, MTS and polyarginine. A negative correlation was observed between the cationic charge on the PTD and the extent of secretion. Poor secretion was found when the PTD charge was greater than +5. One TAT-CRE protein variant had a 14-fold enhancement above CRE alone when added to cells in the presence of chloroquine. This PTD domain also enhanced gene expression after plasmid delivery. These data illustrate that some secreted PTD proteins may be useful reagents to improve protein delivery in mammalian systems and a novel approach to enhancing the response to DNA transfections.
Shaw
Paul A
PA
Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, CB2 3EG, UK.
Catchpole
Ian R
IR
Goddard
Catharine A
CA
Colledge
William H
WH
D000595:Amino Acid Sequence; D020126:Brefeldin A / Q000494:pharmacology; D002412:Cations / Q000737:chemistry; D002460:Cell Line; D045683:Furin / Q000037:antagonists & inhibitors / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D019426:Integrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011480:Protease Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D019076:Transgenes / Q000235:genetics
D002412:Cations; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D020126:Brefeldin A; C045073:Cre recombinase; D019426:Integrases; D045683:Furin