Carboxyl-terminal domain of human apolipoprotein E: expression, purification, and crystallization.
Thioredoxin fusion expression vectors for two carboxyl-terminal fragments of human apolipoprotein (apo) E (residues 223-272 and 223-299) were generated from an apoE cDNA with the objective of obtaining structural information on this functionally important region of apoE by X-ray crystallography. A thrombin cleavage recognition site was positioned at the fusion junction to release the apoE fragments from the fusion protein. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates by nickel-affinity column chromatography, and cleaved with thrombin. After gel filtration and ion exchange chromatography, yields of each fragment were approximately 14 mg/L. Both fragments bind to the phospholipid dimyristoylphosphatidylcholine in a manner similar to that of the 216-299 fragment of apoE isolated from plasma, which represents the major lipid-binding region of the protein. Orthorhombic crystals of the apoE 223-272 fragment that diffracted to 1.8 A were obtained in a mixture of 0.1 M imidazole (pH 6.0) and 0.4 M NaOAc (pH 7.0-7.5), containing 30% glycerol. The space group is C222 with cell dimensions of a = 35.17 A, b = 38.95 A, and c = 133.27 A.
Forstner
M
M
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California, 94550, USA.
Peters-Libeu
C
C
Contreras-Forrest
E
E
Newhouse
Y
Y
Knapp
M
M
Rupp
B
B
Weisgraber
K H
KH
D020816:Amino Acid Motifs; D001057:Apolipoproteins E / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001665:Binding Sites / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013879:Thioredoxins / Q000235:genetics
D001057:Apolipoproteins E; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013879:Thioredoxins