Gi/o proteins: expression for direct activation enquiry.
G protein-mediated pathways are fundamental mechanisms of cell signaling. In this paper, the expression and the characterization of the alphai1, alphai3, alphao1, beta1, and gamma2 subunits of the human G protein are described. This approach was developed to evaluate the G protein activation profile of new compounds. pCR-TOPO T7 vectors, engineered to contain the target sequences, were used to transform Escherichia coli competent cells. Subunits were over-expressed in a preparative scale as fusion proteins with a six-histidine tag, and subsequently purified by metal chelate chromatography. Afterward, the His-tag was removed by enterokinase digestion, and the secondary structures of the recombinant subunits were analyzed by circular dichroism. To assess the functionality of the subunits, the rate of GTP hydrolysis and GTPgammaS binding were evaluated both in the absence and in the presence of two modulators: the peptidic activator Mastoparan and the non-peptidic activator N-dodecyl-lysinamide (ML250). Tests were conducted on isolated alpha-subunit and on heterotrimeric alphabetagamma complex, alone or reconstituted in phospholipidic vesicles. Our results show that recombinant subunits are stable, properly folded and, fully active, which makes them suitable candidates for functional studies.
Di Cesare Mannelli
Lorenzo
L
Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50134 Florence, Italy. lorenzo.mannelli@unifi.it
Pacini
Alessandra
A
Toscano
Annarita
A
Fortini
Martina
M
Berti
Debora
D
Ghelardini
Carla
C
Galeotti
Nicoletta
N
Baglioni
Piero
P
Bartolini
Alessandro
A
D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides / Q000502:physiology; D010957:Plasmids; D014862:Wasp Venoms
D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014862:Wasp Venoms; C037500:mastoparan; D019206:GTP-Binding Protein alpha Subunits, Gi-Go