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Expression of cDNAs for G proteins in Escherichia coli. Two forms of Gs alpha stimulate adenylate cyclase.

Publication ,  Journal Article
Graziano, MP; Casey, PJ; Gilman, AG
Published in: J Biol Chem
August 15, 1987

Complementary DNAs that encode two forms of the alpha subunit (Gs alpha) of the guanine nucleotide-binding protein responsible for stimulation of adenylate cyclase (Gs) have been inserted into plasmid vectors for expression in Escherichia coli. Following transformation of either of these plasmids into E. coli K38, Gs alpha accumulates to 0.4-0.8 mg/liter (approximately 0.1% of total protein), as judged by immunoblot analysis with specific antisera. Based on deduced amino acid sequence, the two cDNAs should encode proteins with molecular weights of 44,500 and 46,000, respectively (Robishaw, J.D., Smigel, M. D., and Gilman, A. G. (1986) J. Biol. Chem. 261, 9587-9590). Expression of these cDNAs in E. coli yields proteins that co-migrate on sodium dodecyl sulfate-polyacrylamide gels with the Gs alpha subunits from S49 lymphoma cell membranes, with apparent molecular weights of 45,000 and 52,000, respectively. Low levels of activity are detected in the 100,000 X g supernatant after lysis and fractionation of E. coli expressing either form of Gs alpha. Partial purification of Gs alpha from E. coli lysates yields preparations in which significant and stable activity can be assayed. Both forms of Gs alpha migrate through sucrose gradients as soluble, monodisperse species in the absence of detergent. As expressed in E. coli, both forms of Gs alpha can reconstitute isoproterenol-, guanine nucleotide-, and fluoride-stimulated adenylate cyclase activity in S49 cyc-cell membranes to approximately the same degree and can be ADP-ribosylated with [32P]NAD+ and cholera toxin. However, based on the specific activity of purified rabbit liver Gs, only 1-2% of the Gs alpha expressed in E. coli appears to be active. Incubation of partially purified fractions of recombinant Gs alpha with guanosine 5'-(3-O-thio)triphosphate and resolved beta gamma subunits isolated from purified bovine brain G proteins results in a 7-10-fold increase in Gs activity. Incubation of bovine brain beta gamma with recombinant Gs alpha also leads to a dramatic increase in observed levels of cholera toxin-catalyzed [32P]ADP-ribosylation.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

August 15, 1987

Volume

262

Issue

23

Start / End Page

11375 / 11381

Location

United States

Related Subject Headings

  • Transformation, Genetic
  • Thionucleotides
  • T-Phages
  • Plasmids
  • Isoproterenol
  • Guanosine Triphosphate
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • GTP-Binding Proteins
  • Escherichia coli
  • DNA-Directed RNA Polymerases
 

Citation

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ICMJE
MLA
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Graziano, M. P., Casey, P. J., & Gilman, A. G. (1987). Expression of cDNAs for G proteins in Escherichia coli. Two forms of Gs alpha stimulate adenylate cyclase. J Biol Chem, 262(23), 11375–11381.
Graziano, M. P., P. J. Casey, and A. G. Gilman. “Expression of cDNAs for G proteins in Escherichia coli. Two forms of Gs alpha stimulate adenylate cyclase.J Biol Chem 262, no. 23 (August 15, 1987): 11375–81.
Graziano MP, Casey PJ, Gilman AG. Expression of cDNAs for G proteins in Escherichia coli. Two forms of Gs alpha stimulate adenylate cyclase. J Biol Chem. 1987 Aug 15;262(23):11375–81.
Graziano, M. P., et al. “Expression of cDNAs for G proteins in Escherichia coli. Two forms of Gs alpha stimulate adenylate cyclase.J Biol Chem, vol. 262, no. 23, Aug. 1987, pp. 11375–81.
Graziano MP, Casey PJ, Gilman AG. Expression of cDNAs for G proteins in Escherichia coli. Two forms of Gs alpha stimulate adenylate cyclase. J Biol Chem. 1987 Aug 15;262(23):11375–11381.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

August 15, 1987

Volume

262

Issue

23

Start / End Page

11375 / 11381

Location

United States

Related Subject Headings

  • Transformation, Genetic
  • Thionucleotides
  • T-Phages
  • Plasmids
  • Isoproterenol
  • Guanosine Triphosphate
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • GTP-Binding Proteins
  • Escherichia coli
  • DNA-Directed RNA Polymerases