Centrifugal Precipitation Chromatography: Principle, Apparatus, and Optimization of Key Parameters for Protein Fractionation by Ammonium Sulfate Precipitation

Ito, Yoichiro

doi:10.1006/abio.1999.4365

Cited by 28 publications

(22 citation statements)

References 8 publications

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“…A particular advantage of the CCC approach is the fact that the system tolerates particulate samples. Therefore, the one-step purification of HRP employing CCC resulted in a simple preparative procedure which may be further scaled-up for industrial separations [21,22].…”

Section: Discussionmentioning

confidence: 99%

Performance of an aqueous two‐phase‐based countercurrent chromatographic system for horseradish peroxidase purification

Magri¹,

Cabrera²,

Miranda³

et al. 2003

J of Separation Science

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Performance of an aqueous two-phase-based countercurrent chromatographic system for horseradish peroxidase purification Countercurrent chromatography (CCC) purification of horseradish peroxidase (HRP) from Armoracia rusticana root extracts was achieved by employing polymer-phosphate aqueous two-phase systems (ATPS). By using preparative columns at 1000 rpm, a 25 -30% retention of the top phase of an ATPS composed of 10% w/w PEG 1540 and 14.8% w/w phosphate -with added 2 mol/kg sodium chloride -was obtained. The retention level was stable during the standard separation running time (4 h). Horseradish root extract samples were injected into the system (10 -25 mL; 200 -250 U/mL peroxidase; 2.0 -4.0 mg/mL total protein). Retention of HRP in the CCC "column" during the chromatographic run was attained in the selected ATPS, where the partition coefficient K for the enzyme was G 8. Replacement of the mobile phase with a fresh one but in the absence of added salt brought about product elution. Recovery of HRP in this fraction accounts for G 45% of the total activity loaded, with a purification factor of 6. Enzyme activity was also found in the pass-through fraction and in the remaining liquid (stationary) phase, a fact that should be ascribed to the existence of multiple peroxidase isoforms. SDS-PAGE of the active fraction showed a protein band at 44 kDa, compatible with the presence of HRP. Thus, the optimised CCC system allowed the separation of HRP directly from a complex biological material. These results open up the possibility of achieving protein separation with CCC/ ATPS and of scaling-up processes in industrial separators.

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Section: Discussionmentioning

confidence: 99%

Performance of an aqueous two‐phase‐based countercurrent chromatographic system for horseradish peroxidase purification

Magri¹,

Cabrera²,

Miranda³

et al. 2003

J of Separation Science

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“…AS precipitation was put at the first step of the purification scheme for its easy operation and economical practicality [11,12]. From Fig.…”

Section: Discussionmentioning

confidence: 99%

Establishment of a three-step purification scheme for a recombinant protein rG17PE38 and its characteristics identification

Xia

Liu

et al. 2015

Journal of Chromatography B

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“…Separation of RNA and plasmid DNA by centrifugal precipitation chromatography was performed in the unit whose separation principle and basic design were described in detail elsewhere (Ito, 2000;Sookkumnerd et al, 2000). The separation column used in the present study was made of a pair of flat disks (high-density polyethylene, 13.2 cm in diameter and 1.5 cm in thickness) with mutually mirrorimaged spiral grooves (1.5 mm wide, 2.0 mm deep, f 2 m long).…”

Section: Centrifugal Precipitation Chromatography Of E Coli Clarifiementioning

confidence: 99%

“…Before starting each experiment, both upper and lower channels were completely filled with 0.30% (w/v) Figure 1. Design of the centrifugal precipitation chromatography (Ito, 2000): (A) a pair of separation disks and dialysis membrane, (B) schematic diagram of elution system of centrifugal precipitation chromatography. CTAB solution.…”

Section: Centrifugal Precipitation Chromatography Of E Coli Clarifiementioning

confidence: 99%

“…This in turn results in solute fractionation by the time they finally elute out at the end of the channel. The CPC system has been successfully applied to the separation of biopolymers such as purification of monoclonal antibody from cell culture supernatant, affinity separation of recombinant ketosteroid isomerase from crude E. coli lysate, and separation of different protein molecules (Ito, 2000). Since the membrane's surface area is much smaller than the total surface area of packed particles in conventional chromatography, product loss caused by the adsorption of biomolecules on solid phase would be minimized in the CPC process (Sookkumnerd et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

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Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH₄Cl

Tomanee¹,

Hsu²,

Ito³

2004

Biotech & Bioengineering

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Centrifugal precipitation chromatography (CPC) is a separation system that mainly employs a moving concentration gradient of precipitating agent along a channel and solutes of interest undergo repetitive precipitation-dissolution, fractionate at different locations, and elute out from the channel according to their solubility in the precipitating agent solution. We report here for the first time the use of a CPC system for fractionation of protein, RNA, and plasmid DNA in clarified lysate produced from bacterial culture. The cationic surfactant cetyltrimethylammonium bromide (CTAB) was initially used as a precipitating agent; however, all biomolecules showed no differential solubility in the moving concentration gradient of this surfactant and, as a result, no separation of protein, RNA, and plasmid DNA occurred. To overcome this problem, inorganic salts such as NaCl and NH(4)Cl were introduced into solution of CTAB. The protein and RNA were found to have higher solubility with the addition of these salts and separated from the plasmid DNA. Decreasing surface charge density of CTAB upon addition of NaCl and NH(4)Cl was believed to lead to lower surfactant complexation, and therefore caused differential solubility and fractionation of these biomolecules. Addition of CaCl(2) did not improve solubility and separation of RNA from plasmid DNA.

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Centrifugal Precipitation Chromatography: Principle, Apparatus, and Optimization of Key Parameters for Protein Fractionation by Ammonium Sulfate Precipitation

Cited by 28 publications

References 8 publications

Performance of an aqueous two‐phase‐based countercurrent chromatographic system for horseradish peroxidase purification

Performance of an aqueous two‐phase‐based countercurrent chromatographic system for horseradish peroxidase purification

Establishment of a three-step purification scheme for a recombinant protein rG17PE38 and its characteristics identification

Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH₄Cl

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Centrifugal Precipitation Chromatography: Principle, Apparatus, and Optimization of Key Parameters for Protein Fractionation by Ammonium Sulfate Precipitation

Cited by 28 publications

References 8 publications

Performance of an aqueous two‐phase‐based countercurrent chromatographic system for horseradish peroxidase purification

Performance of an aqueous two‐phase‐based countercurrent chromatographic system for horseradish peroxidase purification

Establishment of a three-step purification scheme for a recombinant protein rG17PE38 and its characteristics identification

Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH4Cl

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Product

Resources

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Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH₄Cl