A novel process for the large-scale purification of recombinant tick anticoagulant peptide using perfusion chromatography.
Tick anticoagulant peptide (TAP) is a 60 amino acid peptide (Mr = 6977, pI = 4.9) found in the saliva of the soft tick Ornithodorous moubata that specifically inhibits blood coagulation factor Xa (fXa). A recombinant form of TAP (rTAP) secreted by Saccharomyces cerevisiae was purified from 200 liters of fermentation broth with POROS, strong cation exchange (SCX) and reversed-phase high performance liquid perfusion chromatography (RP-HPLC) media (20 microns nominal particle diameter). The higher linear flow rates and dynamic capacities, as well as low back pressures, obtained with perfusion chromatography media permitted 37.5 g of rTAP to be efficiently captured and significantly enriched from 400 liters of diafiltered fermentation broth (24.3 g yield) with 1.25 liter of SCX media using a low pressure column and peristaltic pump in 4.5 hours. Subsequently, 16.7 g of rTAP obtained from the capture step was purified in a high-resolution mode with the same SCX media, after the media was cleaned and repacked into an 800 ml high-pressure column. By doing multiple rapid cycles at high linear flow rates, preparative-scale high-resolution purification of multi-gram amounts of the peptide was done on this relatively small column in only 10.5 hours. Finally, the peptide was desalted and decolorized on a 200 ml RP-HPLC column of perfusion chromatography media by doing multiple rapid cycles. After lyophilization, 12 g of peptide (46.9% yield) was obtained that was > 96% homogeneous by several analytical criteria and fully active in inhibiting blood coagulation factor Xa (fXa).(ABSTRACT TRUNCATED AT 250 WORDS)
Lehman
E D
ED
Department of Cellular and Molecular Biology, Merck Research Laboratories, West Point, PA 19486.
Joyce
J G
JG
Freymeyer
D K
DK
Bailey
F J
FJ
Herber
W K
WK
Miller
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D005285:Fermentation; D005813:Genes, Synthetic; D036341:Intercellular Signaling Peptides and Proteins; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification*; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D013987:Ticks
D060829:Arthropod Proteins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C063967:tick anticoagulant peptide; D000072235:Mating Factor