« Previous AbstractEvidence for early intracellular accumulation of volatile compounds during spadix development in Arum italicum L. and preliminary data on some tropical Aroids    Next AbstractEnhanced in vivo IgE production and T cell polarization toward the type 2 phenotype in association with indoor exposure to VOC: results of the LARS study »

Biotechnology (N Y)

Title:		A novel process for the large-scale purification of recombinant tick anticoagulant peptide using perfusion chromatography
Author(s):		Lehman ED; Joyce JG; Freymeyer DK; Bailey FJ; Herber WK; Miller WJ;
Address:		"Department of Cellular and Molecular Biology, Merck Research Laboratories, West Point, PA 19486"
Journal Title:		Biotechnology (N Y)
Year:		1993
Volume:		11
Issue:		2
Page Number:		207 - 212
DOI:		10.1038/nbt0293-207
ISSN/ISBN:		0733-222X (Print) 0733-222X (Linking)
Abstract:		"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)"
Keywords:		"Amino Acid Sequence Animals Arthropod Proteins *Chromatography, High Pressure Liquid/methods Chromatography, Ion Exchange Fermentation Genes, Synthetic Intercellular Signaling Peptides and Proteins Mating Factor Molecular Sequence Data Peptides/genetics/*;"
Notes:		"MedlineLehman, E D Joyce, J G Freymeyer, D K 2nd Bailey, F J Herber, W K Miller, W J eng 1993/02/01 Biotechnology (N Y). 1993 Feb; 11(2):207-12. doi: 10.1038/nbt0293-207"