Large-scale purification and characterization of recombinant tick anticoagulant peptide

J Chromatogr. 1992 Feb 14;574(2):225-35. doi: 10.1016/0378-4347(92)80034-n.

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.

MeSH terms

  • Amino Acid Sequence
  • Arthropod Proteins
  • Chromatography, High Pressure Liquid
  • Electrophoresis, Polyacrylamide Gel
  • Factor Xa Inhibitors
  • Fermentation
  • Genes, Synthetic
  • Humans
  • Intercellular Signaling Peptides and Proteins
  • Isoelectric Focusing
  • Mass Spectrometry
  • Molecular Sequence Data
  • Peptides / genetics
  • Peptides / isolation & purification*
  • Peptides / metabolism
  • Peptides / pharmacology
  • Plasmids
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacology
  • Saccharomyces cerevisiae / metabolism

Substances

  • Arthropod Proteins
  • Factor Xa Inhibitors
  • Intercellular Signaling Peptides and Proteins
  • Peptides
  • Recombinant Proteins
  • tick anticoagulant peptide