Development and application of centrifugal precipitation chromatography has been continued. The system internally generates a concentration gradient of ammonium sulfate (AS) along a long channel to fractionate proteins according to their solubility in AS solution. The separation column consists of a pair of disks with mutually mirror-imaged spiral channels that are separated by a semipermeable membrane. The disk assembly is mounted on a sealless continuous flow centrifuge. Concentrated As solution is introduced into the upper channel while a water solution is passed through the lower channel in the opposite direction in a rotating column. A mixture of proteins injected into the water channel moves along an AS gradient of increasing concentration that has been established in the water solution. Each protein species precipitates at a different AS concentration along the gradient. The eluate is continuously monitored and collected using a fraction collector. A series of basic experiments was performed to study the rates of AS transfer and osmosis through the membrane, and the operational parameters including elution time,revolution speed, inclination of the gradient and sample size were optimized using stable protein samples. A new set of columns with different channel configuration was made to improve the efficiency of separation. Preliminary applications were successful in purification of monoclonal antibodies from cell culture supernatant and ascitic fluid, and affinity separation of recombinant ketosteroid isomerase from a crude E. coli lysate. The method was also successfully applied to fractionation of other biopolymers such as hyaluronic acid, chondroitin sulfate, polymerized catechins, and dextran using a gradient between suitable organic solvents and water.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL001047-04
Application #
6541679
Study Section
(LBC)
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2001
Total Cost
Indirect Cost
Name
U.S. National Heart Lung and Blood Inst
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Baldermann, Susanne; Fleischmann, Peter; Bolten, Mareike et al. (2009) Centrifugal precipitation chromatography, a powerful technique for the isolation of active enzymes from tea leaves (Camellia sinensis). J Chromatogr A 1216:4263-7
Inui, Masafumi; Fukui, Akimasa; Ito, Yuzuru et al. (2006) Xapelin and Xmsr are required for cardiovascular development in Xenopus laevis. Dev Biol 298:188-200
Cao, Xue-Li; Xu, Ya-Tao; Zhang, Guang-Ming et al. (2006) Purification of coenzyme Q10 from fermentation extract: high-speed counter-current chromatography versus silica gel column chromatography. J Chromatogr A 1127:92-6
Shibusawa, Yoichi; Takeuchi, Naoko; Sugawara, Kazusa et al. (2006) Aqueous-aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins. J Chromatogr B Analyt Technol Biomed Life Sci 844:217-22
Shibusawa, Yoichi; Yamakawa, Yutaka; Noji, Ryoko et al. (2006) Three-phase solvent systems for comprehensive separation of a wide variety of compounds by high-speed counter-current chromatography. J Chromatogr A 1133:119-25
Takeda, Naoya; Kondo, Masashi; Ito, Satoru et al. (2006) Role of RhoA inactivation in reduced cell proliferation of human airway smooth muscle by simvastatin. Am J Respir Cell Mol Biol 35:722-9
Ito, Yoichiro (2005) Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. J Chromatogr A 1065:145-68
Yu, Henry; Ito, Yoichiro (2004) Preparative separation of proteins using centrifugal precipitation chromatography based on solubility in ammonium sulfate solution. Prep Biochem Biotechnol 34:1-12
Ito, Y (2000) Centrifugal precipitation chromatography: principle, apparatus, and optimization of key parameters for protein fractionation by ammonium sulfate precipitation. Anal Biochem 277:143-53