Liposome-mediated cellular delivery of active gp91(phox).
BACKGROUND
Gp91(phox) is a transmembrane protein and the catalytic core of the NADPH oxidase complex of neutrophils. Lack of this protein causes chronic granulomatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill microorganisms.


METHODS
Here we optimize a prokaryotic cell-free expression system to produce integral mammalian membrane proteins.


CONCLUSIONS
Using this system, we over-express truncated forms of the gp91(phox) protein under soluble form in the presence of detergents or lipids resulting in active proteins with a "native-like" conformation. All the proteins exhibit diaphorase activity in the presence of cytosolic factors (p67(phox), p47(phox), p40(phox) and Rac) and arachidonic acid. We also produce proteoliposomes containing gp91(phox) protein and demonstrate that these proteins exhibit activities similar to their cellular counterpart. The proteoliposomes induce rapid cellular delivery and relocation of recombinant gp91(phox) proteins to the plasma membrane. Our data support the concept of cell-free expression technology for producing recombinant proteoliposomes and their use for functional and structural studies or protein therapy by complementing deficient cells in gp91(phox) protein.
Marques
Bruno
B
HumProTher, UMR-CNRS 5525, Université Joseph Fourier, Centre Hospitalier Universitaire, Laboratoire d'Enzymologie/DBPC/BP 217, Centre Hospitalier Universitaire de Grenoble, Grenoble, France.
Liguori
Lavinia
L
Paclet
Marie-Hélène
MH
Villegas-Mendéz
Ana
A
Rothe
Romy
R
Morel
Françoise
F
Lenormand
Jean-Luc
JL
D001483:Base Sequence; D002460:Cell Line; D002474:Cell-Free System; D017931:DNA Primers; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D011510:Proteolipids
D017931:DNA Primers; D008562:Membrane Glycoproteins; D011510:Proteolipids; C020485:proteoliposomes; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D008058:Dihydrolipoamide Dehydrogenase