Expression, characterization and purification of soluble G‐protein βγ dimers composed of defined subunits in baculovirus‐infected insect cells

FEBS Letters

Camps, Montserrat¹  Spicher, Karsten²  Gierschik, Peter¹  Dietrich, Alexander¹  Schultz, Günther²  Meister, Michael¹ 

[1] Abteilung Molekulare Pharmakologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany;Institut für Pharmakologie der Freien Universität Berlin, Berlin, Germany

关键词: G-protein;    Signal transduction: Heterologous expression;    Baculovirus;    Spodoptera frugiperda;    Trichoplasia ni;    G-protein;    signal-transducing heterotrimeric guanine nucleotide-binding protein;    S19 cells;    Spodoptera frugiperda cells;    AcNPV;    Autographa californica nuclear polyhedrosis virus.;   

DOI  :  10.1016/0014-5793(92)81195-R

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Recombinant β₁γ₂ dimers of signal-transducing guanine nucleotide-binding proteins (G-proteins) carrying a mutation known to block isoprenylation of the γ₂ subunit were expressed as a soluble protein in baculovirus-infected insect cells. The soluble βγ dimer was analyzed by sucrose density gradient centrifugation and purified to near homogeneity in the absence of detergents. The sedimentation velocity studies gave an s _20,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 βγ dimers suitable for functional and structural characterization.

【 授权许可】

Unknown