Expression, characterization and purification of soluble G-protein beta gamma dimers composed of defined subunits in baculovirus-infected insect cells.
Recombinant beta 1 gamma 2 dimers of signal-transducing guanine nucleotide-binding proteins (G-proteins) carrying a mutation known to block isoprenylation of the gamma 2 subunit were expressed as a soluble protein in baculovirus-infected insect cells. The soluble beta gamma dimer was analyzed by sucrose density gradient centrifugation and purified to near homogeneity in the absence of detergents. The sedimentation velocity studies gave an S20,w value of 4.1 +/- 0.4 S. The two subunits segregated as a dimer upon sucrose density gradient centrifugation and purification by sequential ion exchange and hydroxylapatite chromatography. The results show that baculovirus-infected insect cells can be employed for high level production of pure G-protein beta gamma dimers suitable for functional and structural characterization.
Dietrich
A
A
Abteilung Molekulare Pharmakologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Meister
M
M
Spicher
K
K
Schultz
G
G
Camps
M
M
Gierschik
P
P
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009036:Moths; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins