EXPRESSION, PURIFICATION AND CHARACTERIZATION OF RECOMBINANT CRAMBIN

Crambin, a small hydrophobic protein (4.7 kDa and 46 residues), has be en successfully expressed in Escherichia coli from an artificial, synt hetic gene, Several expression systems were investigated, Ultimately, crambin was successfully expressed as a fusion protein with the maltos e binding protein, which was purified by affinity chromatography, Cram bin expressed as a C-terminal domain was then cleaved from the fusion protein,vith Factor Xa protease and purified, Circular dichroism spect roscopy and amino acid analysis suggested that the purified material w as identical to crambin isolated from seed, For positive identificatio n the protein was crystallized from an ethanol-water solution, by a no vel method involving the inclusion of phospholipids in the crystalliza tion buffer, and then subjected to crystallographic analysis, Diffract ion data were collected at the Brookhaven synchrotron (beamline-X12C) to a resolution of 1.32 Angstrom at 150 K, The structure, refined to a n R value Of 9.6%, confirmed that the cloned protein was crambin, The availability of cloned crambin will allow site-specific mutagenesis st udies to be performed on the protein known to the highest resolution.