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

Abstract

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.

Isozyme-specific Stimulation of Phospholipase C-γ2 by Rac GTPases

“…Miscellaneous-Recombinant baculoviruses were produced as described (43). A baculovirus-encoding Escherichia coli ␤-galactosidase (44) was a gift from Dr. Michael Ruffing.…”

Stimulation of phospholipase C‐β2 by recombinant guanine‐nucleotide‐binding protein βγ dimers produced in a baculovirus/insect cell expression system

“…The correct orientation of the inserts to the polyhedrin promoter was verified by DNA sequencing. The cDNA of the human subunit was generated as previously described [33] and ligated into the unique BglII site of the vector pAcUW51 already carrying the cDNA of y1 or y,C71S. The correct orientation of the inserts to the p10 promoter was verified by DNA sequencing.…”

“…We have recently reported the expression, characterization and purification of soluble p,y2C68S dimers in baculovirus-infected insect cells [33]. Our failure to reconstitute this p y preparation with phospholipase C prompted us to characterize the relevance of the carboxy-terminal y subunit isoprenylation to py subunit stimulation of phospholipase C in more detail.…”

Identification of a Three‐Amino‐Acid Region in G Protein γ1 as a Determinant of Selective βγ Heterodimerization

“…The cDNA encoding the human /3, subunit was generated as previously described [19]. The cDNA of human p2 was PCRamplified using synthetic oligonucleotides complementary to the ends of the coding region and carrying recognition sites for EcoRI and BamHI on the 5' and 3' end, respectively.…”

“…Sucrose density gradient centrifugation. Sucrose density gradient centrifugation was performed as described in [19], except that 100 pl of the soluble fraction of baculovirus-infected Trichoplusia ni cells (2 mg protein) was combined with SO p1 buffer A ((20 mM Tris/HCI pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 3 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 10 pM leupeptin, and 2 pg/ml trypsin inhibitor) containing 0.5 mg cytochrome c and applied to the top of the gradient. After centrifugation, the bottom of the tubes were punctured and fractions of six drops each were collected.…”