Delivery of Therapeutic Proteins as Secretable TAT Fusion Products

TY - JOUR

T1 - Delivery of Therapeutic Proteins as Secretable TAT Fusion Products

AU - Flinterman, Marcella

AU - Farzaneh, Farzin

AU - Habib, Nagy

AU - Malik, Farooq

AU - Gaken, Joop

AU - Tavassoli, Mahvash

N1 - joint last author

PY - 2009

Y1 - 2009

N2 - The trans-acting activator of transcription (TAT) protein transduction domain (PTD) mediates the transduction of peptides and proteins into target cells. The TAT-PTD has an important potential as a tool for the delivery of therapeutic agents. The production of TAT fusion proteins in bacteria, however, is problematic because of protein insolubility and the absence of eukaryotic post-translational modification. An attractive alternative, both for in vitro protein production and for in vivo applications, is the use of higher eukaryotic cells for secretion of TAT fusion proteins. However, the ubiquitous expression of furin endoprotease (PACE or SPC1) in the Golgi/endoplasmic reticulum, and the presence of furin recognition sequences within TAT-PTD, results in the cleavage and loss of the TAT-PTD domain during its secretory transition through the endoplasmic reticulum and Golgi. In this study, we show the development of a synthetic TAT kappa-PTD in which mutation of the furin recognition sequences, but retention of protein transduction activity, allows secretion of recombinant proteins, followed by successful uptake of the modified protein, by the target cells. This system was used to successfully secrete marker protein, green fluorescent protein (GFP), and apoptin, a protein with tumor-specific cytotoxicity. Detection of GFP, phosphorylation, and induction of cell death by TAT kappa-GFP-apoptin indicated that the secreted proteins were functional in target cells. This novel strategy therefore has important potential for the efficient delivery of therapeutic proteins.

AB - The trans-acting activator of transcription (TAT) protein transduction domain (PTD) mediates the transduction of peptides and proteins into target cells. The TAT-PTD has an important potential as a tool for the delivery of therapeutic agents. The production of TAT fusion proteins in bacteria, however, is problematic because of protein insolubility and the absence of eukaryotic post-translational modification. An attractive alternative, both for in vitro protein production and for in vivo applications, is the use of higher eukaryotic cells for secretion of TAT fusion proteins. However, the ubiquitous expression of furin endoprotease (PACE or SPC1) in the Golgi/endoplasmic reticulum, and the presence of furin recognition sequences within TAT-PTD, results in the cleavage and loss of the TAT-PTD domain during its secretory transition through the endoplasmic reticulum and Golgi. In this study, we show the development of a synthetic TAT kappa-PTD in which mutation of the furin recognition sequences, but retention of protein transduction activity, allows secretion of recombinant proteins, followed by successful uptake of the modified protein, by the target cells. This system was used to successfully secrete marker protein, green fluorescent protein (GFP), and apoptin, a protein with tumor-specific cytotoxicity. Detection of GFP, phosphorylation, and induction of cell death by TAT kappa-GFP-apoptin indicated that the secreted proteins were functional in target cells. This novel strategy therefore has important potential for the efficient delivery of therapeutic proteins.

U2 - 10.1038/mt.2008.256

DO - 10.1038/mt.2008.256

M3 - Article

SN - 1525-0024

VL - 17

SP - 334

EP - 342

JO - Molecular Therapy

JF - Molecular Therapy

IS - 2

ER -