Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1993 Mar;101(3):713–728. doi: 10.1104/pp.101.3.713

Cloning, expression, and crystallization of jack bean (Canavalia ensiformis) canavalin.

J D Ng 1, T P Ko 1, A McPherson 1
PMCID: PMC158684  PMID: 8310055

Abstract

Canavalin is the major storage protein of the jack bean (Canavalia ensiformis) and belongs to the classical vicilin fraction. A full-length cDNA for canavalin was generated by the polymerase chain reaction. The nucleotide sequence coding for canavalin and the corresponding amino acid sequence were determined and shown to be homologous with those of other seed storage proteins. The amino acid sequence contained an internal sequence duplication corresponding to the structural redundancy in the monomer demonstrated by crystallographic analysis. The coding region of the canavalin cDNA was inserted into a T7 RNA polymerase expression vector and used to transform Escherichia coli. A recombinant protein with a molecular mass of 47 kilodaltons was expressed and purified to 95% homogeneity. The protein exhibited the same physical, immunological, and biochemical properties as native jack bean canavalin. Recombinant canavalin, following treatment with trypsin, was crystallized in two forms. Crystals of a rhombohedral habit grew to 1 mm in the longest dimension and diffracted to beyond 3-A resolution. Three-dimensional diffraction data demonstrated crystals of the recombinant protein to be isomorphous with crystals of the natural plant protein, thereby confirming the identity of their structures.

Full Text

The Full Text of this article is available as a PDF (5.2 MB).

Selected References

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

  1. Birnboim H. C. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983;100:243–255. doi: 10.1016/0076-6879(83)00059-2. [DOI] [PubMed] [Google Scholar]
  2. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  3. Dure L., 3rd An unstable domain in the vicilin genes of higher plants. New Biol. 1990 May;2(5):487–493. [PubMed] [Google Scholar]
  4. Gibbs P. E., Strongin K. B., McPherson A. Evolution of legume seed storage proteins--a domain common to legumins and vicilins is duplicated in vicilins. Mol Biol Evol. 1989 Nov;6(6):614–623. doi: 10.1093/oxfordjournals.molbev.a040575. [DOI] [PubMed] [Google Scholar]
  5. Granger-Schnarr M., Lloubes R., de Murcia G., Schnarr M. Specific protein-DNA complexes: immunodetection of the protein component after gel electrophoresis and Western blotting. Anal Biochem. 1988 Oct;174(1):235–238. doi: 10.1016/0003-2697(88)90540-4. [DOI] [PubMed] [Google Scholar]
  6. Hennessey J. P., Jr, Johnson W. C., Jr Information content in the circular dichroism of proteins. Biochemistry. 1981 Mar 3;20(5):1085–1094. doi: 10.1021/bi00508a007. [DOI] [PubMed] [Google Scholar]
  7. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  8. Lim H. M., Pène J. J. Optimal conditions for supercoil DNA sequencing with the Escherichia coli DNA polymerase I large fragment. Gene Anal Tech. 1988 Mar-Apr;5(2):32–39. doi: 10.1016/0735-0651(88)90024-6. [DOI] [PubMed] [Google Scholar]
  9. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  10. McPherson A., Jr, Rich A. X-ray crystallographic study of the quaternary structure of canavalin. J Biochem. 1973 Jul;74(1):155–160. doi: 10.1093/oxfordjournals.jbchem.a130218. [DOI] [PubMed] [Google Scholar]
  11. McPherson A., Jr, Spencer R. Preliminary structure analysis of canavalin from jack bean. Arch Biochem Biophys. 1975 Aug;169(2):650–661. doi: 10.1016/0003-9861(75)90209-x. [DOI] [PubMed] [Google Scholar]
  12. Mohanty J. G., Elazhary Y. Purification of IgG from serum with caprylic acid and ammonium sulphate precipitation is not superior to ammonium sulphate precipitation alone. Comp Immunol Microbiol Infect Dis. 1989;12(4):153–160. doi: 10.1016/0147-9571(89)90064-7. [DOI] [PubMed] [Google Scholar]
  13. Morrison R., Delozier G., Robinson L., McPherson A. Biochemical and x-ray diffraction analysis of concanavalin B crystals from jack bean. Plant Physiol. 1984 Sep;76(1):175–183. doi: 10.1104/pp.76.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sebastiani F. L., Farrell L. B., Vasquez M., Beachy R. N. Conserved amino acid sequences among plant proteins sorted to protein bodies and plant vacuoles. Can they play a role in protein sorting? Eur J Biochem. 1991 Jul 15;199(2):441–450. doi: 10.1111/j.1432-1033.1991.tb16142.x. [DOI] [PubMed] [Google Scholar]
  15. Smith S. C., Johnson S., Andrews J., McPherson A. Biochemical characterization of canavalin, the major storage protein of jack bean. Plant Physiol. 1982 Oct;70(4):1199–1209. doi: 10.1104/pp.70.4.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  17. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  18. Valls L. A., Winther J. R., Stevens T. H. Yeast carboxypeptidase Y vacuolar targeting signal is defined by four propeptide amino acids. J Cell Biol. 1990 Aug;111(2):361–368. doi: 10.1083/jcb.111.2.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Yamauchi D., Nakamura K., Asahi T., Minamikawa T. cDNAs for canavalin and concanavalin A from Canavalia gladiata seeds. Nucleotide sequence of cDNA for canavalin and RNA blot analysis of canavalin and concanavalin A mRNAs in developing seeds. Eur J Biochem. 1988 Jan 4;170(3):515–520. doi: 10.1111/j.1432-1033.1988.tb13730.x. [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES