Expression, purification and characterization of recombinant crambin

Crambin, a small hydrophobic protein (4.7 kDa and 46 residues),has been successfully expressed in Escherichia coli from anartificial, synthetic gene. Several expression systems wereinvestigated. Ultimately, crambin was successfully expressedas a fusion protein with the maltose binding protein, whichwas purified by affinity chromatography. Crambin expressed asa C-terminal domain was then cleaved from the fusion proteinwith Factor Xa protease and purified. Circular dichroism spectroscopyand amino acid analysis suggested that the purified materialwas identical to crambin isolated from seed. For positive identificationthe protein was crystallized from an ethanol–water solution,by a novel method involving the inclusion of phospholipids inthe crystallization buffer, and then subjected to crystallographicanalysis, Diffraction data were collected at the Brookhavensynchrotron (beamline-X12C) to a resolution of 1.32 Åat 150 K. The structure, refined to an R value of 9.6%, confirmedthat the cloned protein was crambin. The availability of clonedcrambin will allow site-specific mutagenesis studies to be performedon the protein known to the highest resolution.