| 摘要 |
The invention is a method for purifying recombinantly produced TAP, a 60 amino acid anticoagulant protein derived from ticks. S. cerevisiae ATCC 20984 yeast cells are cultured in yeast cell culture media suitable for expressing TAP. The cells are separated from expressed protein by crossflow ultrafiltration and diafiltration to clarify the broth. Preferably, pH is thereafter adjusted to about 3.5. The TAP is then concentrated and partially purified by adsorption on to a POROS cation-exchange resin, preferably a POROS II HS/P sulfopropyl cation-exchange resin. The broth is preferably pumped onto the resin using a peristaltic pump. The protein is eluted from the resin with a concentrated salt buffer, preferably 1 M NaCl. The TAP in the eluate is then purified to greater than 96 % homogeneity with a step gradient generated with a preparative HPLC pump. The protein is then desalted and concentrated on a reverse phase chromatography column, preferably a POROS R/M reverse phase chromatography column, and thereafter lyophilized. |
