Baculovirus Expression and Purification of a Soluble, Mutant G-Protein α Subunit

T. L Z Jones, C. Woodard, Allen M. Spiegel

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The cDNA for the αi1 protein that had undergone site-directed mutagenesis to change glycine-2 to alanine was ligated into a baculovirus transfer vector. A recombinant virus was obtained by transfecting Sf9 cells with both the wild-type baculovirus DNA and the transfer vector and screening for recombinant plaques. Infection with the recombinant virus led to a high level of expression of the mutated αi1 protein in the soluble fraction of the cell. The protein was purified by ammonium sulfate precipitation, gel filtration, and immobilized dye chromatography. The typical yield was 7.5 mg from two 800-ml cultures. The protein showed immunoreactivity to three different αi-specific antibodies. It bound guanosine 5′-(γ-thio)triphosphate (GTPγS) with a stoichiometry of 0.63 to 0.91 mol/mol and with a rate constant (kGTPγS) of binding of 0.126 min-1. When GTPγS bound, the protein was protected from complete tryptic cleavage. The recombinant protein was able to undergo pertussis toxin-catalyzed ADP ribosylation and bind βγ subunits but with a reduced affinity compared to that of a transducin. Thus using a recombinant baculovirus, a nonmyristylated G protein α subunit was abundantly expressed in Sf9 cells and milligram quantities of a functional protein were easily purified.

Original languageEnglish (US)
Pages (from-to)64-71
Number of pages8
JournalProtein Expression and Purification
Volume4
Issue number1
DOIs
StatePublished - Feb 1993
Externally publishedYes

Fingerprint

Baculoviridae
Protein Subunits
Mutant Proteins
GTP-Binding Proteins
Purification
Sf9 Cells
Proteins
Viruses
Transducin
Mutagenesis
Guanosine
Pertussis Toxin
Ammonium Sulfate
Site-Directed Mutagenesis
Chromatography
Recombinant Proteins
Stoichiometry
Alanine
Glycine
Adenosine Diphosphate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Baculovirus Expression and Purification of a Soluble, Mutant G-Protein α Subunit. / Jones, T. L Z; Woodard, C.; Spiegel, Allen M.

In: Protein Expression and Purification, Vol. 4, No. 1, 02.1993, p. 64-71.

Research output: Contribution to journalArticle

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