High-level expression in Escherichia coli of a chemically synthesized gene for [Leu-28]echistatin.
A gene (Ecs) encoding a platelet aggregation inhibitor, echistatin (Ecs), has been chemically synthesized. Met at position 28 of the native protein was replaced by Leu in the recombinant Ecs. To express this synthetic gene in Escherichia coli, an expression vector, pJC264, was constructed by inserting portions of the E. coli cheB and cheY gene complex into the plasmid pUC13. High-level expression of the synthetic [Leu-28]Ecs was achieved by its fusion with the E. coli cheY gene in the expression vector. Recombinant [Leu-28]Ecs was liberated from the fusion protein by CNBr cleavage at the Met inserted between the CheY protein and [Leu-28]Ecs. The recombinant [Leu-28]Ecs was purified to homogeneity by reverse-phase high-performance liquid chromatography. The refolded [Leu-28]Ecs was identical to native Ecs in inhibiting platelet aggregation, suggesting that Met at position 28 is not essential for the biological activity of this platelet aggregation inhibitor.
Gan
Z R
ZR
Department of Biological Chemistry, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Condra
J H
JH
Gould
R J
RJ
Zivin
R A
RA
Bennett
C D
CD
Jacobs
J W
JW
Friedman
P A
PA
Polokoff
M A
MA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005813:Genes, Synthetic; D036341:Intercellular Signaling Peptides and Proteins; D007930:Leucine / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides; D010957:Plasmids; D010974:Platelet Aggregation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D014757:Viper Venoms / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014757:Viper Venoms; C057986:echistatin; D004247:DNA; D007930:Leucine