Expression of human vasopressin and oxytocin receptors in Escherichia coli.
In order to produce large amounts of human vasopressin and oxytocin receptors compatible with direct structural biology approaches such as X-ray crystallography, NMR or mass spectrometry, we have expressed these neurohypophysial hormone receptors in Escherichia coli. To facilitate the level of expression, the coding sequence for the V1a vasopressin receptor and the oxytocin receptor were first optimized for bacterial expression. The resulting 'bacterial receptor cDNAs' were then subcloned into pET/T7-driven prokaryotic expression vectors. Different constructs have been prepared: each cDNA was incorporated alone or in fusion with a T7 tag sequence or a glutathione-S-transferase tag sequence at the N-terminus end. Moreover, a 6 x His tag sequence has been added at the C-terminus end for one-step purification of the receptors. Screening of BL21(DE3) and BL21(DE3)pLysS bacterial strains transformed with the different constructions was achieved by Coomassie blue-stained SDS-polyacrylamide gels and by 6 x His antibody Western blotting. Several clones were selected for purification of the receptors. Expression levels of the receptors are now encouraging and will be optimized for further structural and functional studies. Moreover, at the same time, the construction of the bacterial-optimized sequence of the V2 vasopressin receptor and its expression will be performed.
Mouillac
Bernard
B
INSERM U469, 141 rue de la Cardonille, 34094 Montpellier, France. mouillac@u469.montp.inserm.fr
Sen
Tuhinadri
T
Durroux
Thierry
T
Gaibelet
Gérald
G
Barberis
Claude
C
D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018360:Crystallography, X-Ray; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D018045:Receptors, Oxytocin / Q000737:chemistry* / Q000235:genetics*; D017483:Receptors, Vasopressin / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry
D018045:Receptors, Oxytocin; D017483:Receptors, Vasopressin; D011994:Recombinant Proteins