Cloning, expression, and crystallization of jack bean (Canavalia ensiformis) canavalin.
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.
Ng
J D
JD
Department of Biochemistry, University of California, Riverside 92521.
Ko
T P
TP
McPherson
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004247:DNA; D004926:Escherichia coli; D007887:Fabaceae / Q000235:genetics* / Q000378:metabolism; D015151:Immunoblotting; D008027:Light; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012542:Scattering, Radiation
D010940:Plant Proteins; D011994:Recombinant Proteins; C006882:canavalin protein, Canavalia ensiformis; D004247:DNA