Cloning , Expression , and Crystallization of Jack Bean ( Canava / ia ensiformis ) Canavalin '


Canavalin is the major storage protein of the jack bean (Canavalia ensiformis) and belongs to the classical vicilin fraction. A fulllength 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 interna1 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 microbes, Sumner and Howell subsequently succeeded in reproducing the crystals from sterile solutions by the direct addition of trypsin, chymotrypsin, and severa1 other common proteases. Sumner conjectured that the crystalline protein resulted from the hydrolysis of contaminating proteins that obstructed canavalin crystallization, and he also suggested that the crystals might be a proteolytic product of the native protein. At the time, however, he could not discriminate between the two possibilities. No biological function was ascribed to canavalin, and its biochemical features remained . . 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 grewe to 1 mm in the longest dimension and diffraded 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. unknown. Canavalin next appeared in the literature in 1974, when researchers at the Massachusetts Institute of Technology reproduced Sumner and Howell's earlier work and obtained large crystalline specimens suitable for x-ray diffraction study. In addition, they determined Some of the biochemical properties of canavalin (McPherson and 1973). Later work (Smith et al., 1982) demonstrated that the native protein isolated from the plant had a monomer mbl wt of about 47,000 and that the monomer was cleaved roughly in half by proteases. They further showed that three cleaved but intact monomers were organized about a perfect 3-fold axis of symmetry in a native molecule having a mo1 wt of about 142,000. Not readily explained was x-ray evidence requiring that the two frapents composing a canavalin mOnOmer be virtually identical in a stmctural sense to produce the exceptional pseudo-32 symmetry of the rhombohedral crystals ( M ~ Pherson and Ri&, 1973; McPherson and Spencer, 1975). ~ l , i ~ was perplexing because the two fragments composing the canavalin monomer were clearly derived from the amino and carboxyl terminal halves of an intact subunit. The implication, realized early on, was that the canavalin subunit of M, = 47,000 consisted of two nearly identical structural domains related to one another by a quasi-dyad axis of symmeby. During the course of studies on other plant seed proteins (Johnson et al., 1982), we became aware that the biorhemical properties of canavalin were very similar to those of phaseolin. Exceptions were that phaseolin was glycosylated and composed of mixtures of three different but highly similar subunits, whereas canavalin had no carbohydrate and appeared to be composed of only a single kind of polypeptide (Smith et al., 1982). From this point it became clear that The name canavalin was given in 1919 by the renowned biochemist J.B. Sumner to an amorphous protein precipitate that he had isolated from defatted meal of the jack bean (Canavalia ensiformis). In the Same PaPer (Sumnert 1919)j he also described two other proteins that he had not only purified but crystallized; these were COnCanaValin A and concanavalin B. Both concanavalin A and B were subsequentlY characterized bY a number of techniques and the three-dimensional structures of both Of these proteins are now known bY X-raY diffraction (Reeke et ale, 1975; ~ o m s o n et al., 1984; McPherson, unpublished). In spite of repeated attempts, Sumner himself was unable to crystallize canavalin and it was othenvise given little attention. In 1936, however, S. Howell, a student working in Sumner's laboratory, k f t a sohtion Of CanaValin for an extended period of time on the benchtop without regard for sterility. He subsequently discovered masses of large rhombohedral crystals on the bottom of the flask. He had crystallized canavalin (Sumner and Howell, 1936). Suspecting that the crystallization had somehow been affected by the action of degradative enzymes produced by

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@inproceedings{Bean2002CloningE, title={Cloning , Expression , and Crystallization of Jack Bean ( Canava / ia ensiformis ) Canavalin '}, author={J. N. Bean}, year={2002} }