title: Large-scale purification and characterization of recombinant tick anticoagulant peptide.
abstract: 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.
authors.affiliation: Department of Cellular and Molecular Biology, Merck, Sharp & Dohme Research Laboratories, West Point, PA 19486.
authors.forename: E D
authors.initials: ED
authors.lastname: Lehman
authors.forename: T F
authors.initials: TF
authors.lastname: Schaefer
authors.forename: C T
authors.initials: CT
authors.lastname: Przysiecki
authors.forename: J G
authors.initials: JG
authors.lastname: Joyce
authors.forename: F J
authors.initials: FJ
authors.lastname: Bailey
authors.forename: C A
authors.initials: CA
authors.lastname: Schulman
authors.forename: C J
authors.initials: CJ
authors.lastname: Burke
authors.forename: H G
authors.initials: HG
authors.lastname: Ramjit
authors.forename: W J
authors.initials: WJ
authors.lastname: Miller
mesh_terms: 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
chemical_list: D060829:Arthropod Proteins; D065427:Factor Xa Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011994:Recombinant Proteins; C063967:tick anticoagulant peptide