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 an artificial, synthetic gene. Several expression systems were investigated. Ultimately, crambin was successfully expressed as a fusion protein with the maltose binding protein, which was purified by affinity chromatography. Crambin expressed as a C-terminal domain was then cleaved from the fusion protein with Factor Xa protease and purified. Circular dichroism spectroscopy and amino acid analysis suggested that the purified material was identical to crambin isolated from seed. For positive identification the protein was crystallized from an ethanol-water solution, by a novel method involving the inclusion of phospholipids in the crystallization buffer, and then subjected to crystallographic analysis. Diffraction data were collected at the Brookhaven synchrotron (beamline-X12C) to a resolution of 1.32 A at 150 K. The structure, refined to an R value of 9.6%, confirmed that the cloned protein was crambin. The availability of cloned crambin will allow site-specific mutagenesis studies to be performed on the protein known to the highest resolution.
Lobb
L
L
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, MA 02167, USA.
Stec
B
B
Kantrowitz
E K
EK
Yamano
A
A
Stojanoff
V
V
Markman
O
O
Teeter
M M
MM
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005813:Genes, Synthetic; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D012995:Solubility
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; C024934:crambin protein, Crambe abyssinica