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Expression, characterization and purification of soluble G‐protein βγ dimers composed of defined subunits in baculovirus‐infected insect cells
Author(s) -
Dietrich Alexander,
Meister Michael,
Spicher Karsten,
Schultz Günther,
Camps Montserrat,
Gierschik Peter
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)81195-r
Subject(s) - insect , baculoviridae , protein subunit , microbiology and biotechnology , chemistry , biochemistry , characterization (materials science) , protein expression , biology , spodoptera , gene , recombinant dna , botany , materials science , nanotechnology
Recombinant β 1 γ 2 dimers of signal‐transducing guanine nucleotide‐binding proteins (G‐proteins) carrying a mutation known to block isoprenylation of the γ 2 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.