PLANT PHYSIOLOGY , Vol 101, Issue 3 713-728, Copyright © 1993 by American Society of Plant Biologists
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MOLECULAR BIOLOGY AND GENE REGULATION |
Cloning, Expression, and Crystallization of Jack Bean (Canavalia ensiformis) Canavalin
J. D. Ng, T. P. Ko and A. McPherson
Department of Biochemistry, University of California, Riverside, California 92521
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.
