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. 1988 May 1;251(3):857–864. doi: 10.1042/bj2510857

Isolation and expression of a pea vicilin cDNA in the yeast Saccharomyces cerevisiae.

M D Watson 1, N Lambert 1, A Delauney 1, J N Yarwood 1, R R Croy 1, J A Gatehouse 1, D J Wright 1, D Boulter 1
PMCID: PMC1149081  PMID: 3046604

Abstract

A cDNA clone containing the complete coding sequence for vicilin from pea (Pisum sativum L.) was isolated. It specifies a 50,000-Mr protein that in pea is neither post-translationally processed nor glycosylated. The cDNA clone was expressed in yeast from a 2 micron plasmid by using the yeast phosphoglycerate kinase promoter and initiator codon. The resultant fusion protein, which contains the first 16 amino acid residues of phosphoglycerate kinase in addition to the vicilin sequence, was purified and subsequently characterized. It has slightly slower mobility on SDS/polyacrylamide-gel electrophoresis than standard pea vicilin and forms a mixture of multimers, some of which resemble the native protein.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bacon J. R., Lambert N., Phalp M., Plumb G. W., Wright D. J. Resolution of pea legumin subunits by high-performance liquid chromatography. Anal Biochem. 1987 Jan;160(1):202–210. doi: 10.1016/0003-2697(87)90631-2. [DOI] [PubMed] [Google Scholar]
  2. Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
  3. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen C. Y., Oppermann H., Hitzeman R. A. Homologous versus heterologous gene expression in the yeast, Saccharomyces cerevisiae. Nucleic Acids Res. 1984 Dec 11;12(23):8951–8970. doi: 10.1093/nar/12.23.8951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cramer J. H., Lea K., Slightom J. L. Expression of phaseolin cDNA genes in yeast under control of natural plant DNA sequences. Proc Natl Acad Sci U S A. 1985 Jan;82(2):334–338. doi: 10.1073/pnas.82.2.334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dobson M. J., Tuite M. F., Roberts N. A., Kingsman A. J., Kingsman S. M., Perkins R. E., Conroy S. C., Fothergill L. A. Conservation of high efficiency promoter sequences in Saccharomyces cerevisiae. Nucleic Acids Res. 1982 Apr 24;10(8):2625–2637. doi: 10.1093/nar/10.8.2625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Domoney C., Casey R. Measurement of gene number for seed storage proteins in Pisum. Nucleic Acids Res. 1985 Feb 11;13(3):687–699. doi: 10.1093/nar/13.3.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gatehouse J. A., Lycett G. W., Croy R. R., Boulter D. The post-translational proteolysis of the subunits of vicilin from pea (Pisum sativum L.). Biochem J. 1982 Dec 1;207(3):629–632. doi: 10.1042/bj2070629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gatehouse J. A., Lycett G. W., Delauney A. J., Croy R. R., Boulter D. Sequence specificity of the post-translational proteolytic cleavage of vicilin, a seed storage protein of pea (Pisum sativum L.). Biochem J. 1983 May 15;212(2):427–432. doi: 10.1042/bj2120427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grunstein M., Wallis J. Colony hybridization. Methods Enzymol. 1979;68:379–389. doi: 10.1016/0076-6879(79)68027-8. [DOI] [PubMed] [Google Scholar]
  11. Holland M. J., Holland J. P. Isolation and identification of yeast messenger ribonucleic acids coding for enolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. Biochemistry. 1978 Nov 14;17(23):4900–4907. doi: 10.1021/bi00616a007. [DOI] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Lycett G. W., Delauney A. J., Gatehouse J. A., Gilroy J., Croy R. R., Boulter D. The vicilin gene family of pea (Pisum sativum L.): a complete cDNA coding sequence for preprovicilin. Nucleic Acids Res. 1983 Apr 25;11(8):2367–2380. doi: 10.1093/nar/11.8.2367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Maat J., Smith A. J. A method for sequencing restriction fragments with dideoxynucleoside triphosphates. Nucleic Acids Res. 1978 Dec;5(12):4537–4545. doi: 10.1093/nar/5.12.4537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mellor J., Dobson M. J., Roberts N. A., Kingsman A. J., Kingsman S. M. Factors affecting heterologous gene expression in Saccharomyces cerevisiae. Gene. 1985;33(2):215–226. doi: 10.1016/0378-1119(85)90096-4. [DOI] [PubMed] [Google Scholar]
  16. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  17. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schatz G. Biogenesis of yeast mitochondria: synthesis of cytochrome c oxidase and cytochrome c. Methods Enzymol. 1979;56:40–50. doi: 10.1016/0076-6879(79)56007-8. [DOI] [PubMed] [Google Scholar]
  19. Scott J. H., Schekman R. Lyticase: endoglucanase and protease activities that act together in yeast cell lysis. J Bacteriol. 1980 May;142(2):414–423. doi: 10.1128/jb.142.2.414-423.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Seif I., Khoury G., Dhar R. A rapid enzymatic DNA sequencing technique: determination of sequence alterations in early simian virus 40 temperature sensitive and deletion mutants. Nucleic Acids Res. 1980 May 24;8(10):2225–2240. doi: 10.1093/nar/8.10.2225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Smith R. A., Duncan M. J., Moir D. T. Heterologous protein secretion from yeast. Science. 1985 Sep 20;229(4719):1219–1224. doi: 10.1126/science.3939723. [DOI] [PubMed] [Google Scholar]
  22. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tuite M. F., Dobson M. J., Roberts N. A., King R. M., Burke D. C., Kingsman S. M., Kingsman A. J. Regulated high efficiency expression of human interferon-alpha in Saccharomyces cerevisiae. EMBO J. 1982;1(5):603–608. doi: 10.1002/j.1460-2075.1982.tb01215.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Utsumi S., Mori T. Studies on subunit assembly of broad bean legumin in a reconstitution system. J Biochem. 1983 Dec;94(6):2001–2008. doi: 10.1093/oxfordjournals.jbchem.a134554. [DOI] [PubMed] [Google Scholar]
  25. Zaret K. S., Sherman F. DNA sequence required for efficient transcription termination in yeast. Cell. 1982 Mar;28(3):563–573. doi: 10.1016/0092-8674(82)90211-2. [DOI] [PubMed] [Google Scholar]

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