Large-scale purification and characterization of recombinant tick anticoagulant peptide.
Recombinant tick anticoagulant peptide (r-TAP), a potent and specific inhibitor of blood coagulation factor Xa, was purified to greater than 99% homogeneity at the multi-gram scale. Genetically engineered yeast secreted 200-250 mg/l of the heterologous protein into the medium. Cells were separated from broth by diafiltration and purification was done by two chromatographic steps, both conducive to operation on a large scale. Analysis of the purified protein by several methods indicated that it was greater than 99% homogeneous and no incompletely processed or truncated proteins were detected. Physico-chemical characterization data of r-TAP show that it exists as a monomer in solution and no evidence of post-translational modification was observed. The purified protein was fully active in inhibiting human coagulation factor Xa.
Lehman
E D
ED
Department of Cellular and Molecular Biology, Merck, Sharp & Dohme Research Laboratories, West Point, PA 19486.
Schaefer
T F
TF
Przysiecki
C T
CT
Joyce
J G
JG
Bailey
F J
FJ
Schulman
C A
CA
Burke
C J
CJ
Ramjit
H G
HG
Miller
W J
WJ
D000595:Amino Acid Sequence; D060829:Arthropod Proteins; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D065427:Factor Xa Inhibitors; D005285:Fermentation; D005813:Genes, Synthetic; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D060829:Arthropod Proteins; D065427:Factor Xa Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011994:Recombinant Proteins; C063967:tick anticoagulant peptide