Purification of Glucosyltransferase from<i>Streptococcus sobrinus</i>Cell Culture Medium by Combined Use of Batch Extraction and Countercurrent Chromatography with a Polymer Phase System

Shibusawa, Yöichi; Isozaki, Mitsuhiro; Yanagida, Akio; Shindo, Heisaburo; Ito, Yoichiro

doi:10.1081/jlc-200025713

Cited by 4 publications

(3 citation statements)

References 28 publications

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“…Therefore, it is necessary to remove both PEG 3350 and dextran T40 before application to the gel. In our previous papers [21,22] dealing with GTF purification by CCC using aqueous polymer two-phase systems, we have used high molecular weight polymers of PEG 8000 (Mr: 6000-7500) and dextran T500 (Mr: 500,000). After the purification of GTF by CCC, the CCC fractions were diluted with 10-fold volume of potassium phosphate buffer and passed through a hydroxylapatite column to eliminated these polymers.…”

Section: Removal Of Peg and Dextran From CCC Fractions By Ultrafiltramentioning

confidence: 99%

“…In the past the CCC separations of basic histones, serum proteins [20], and profilin-actin complex from crude Acanthamoeba extract [18] were performed using PEG-dextran two-phase systems. Recently, we have demonstrated the purification of glucosyltransferase (GTF) from S. mutans cell-lysate and S. sobrinus culture medium [21,22] using a 7.7% PEG 8000-4.0% dextran T500 solvent system. After CCC purification, these polymers were removed from CCC fractions by hydroxyapatite chromatography.…”

Section: Introductionmentioning

confidence: 99%

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Aqueous–aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins

Shibusawa

Takeuchi

Sugawara

et al. 2006

Journal of Chromatography B

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Section: Removal Of Peg and Dextran From CCC Fractions By Ultrafiltramentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Aqueous–aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins

Shibusawa

Takeuchi

Sugawara

et al. 2006

Journal of Chromatography B

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“…In the past the CCC separations of basic histones, serum proteins [25], and profilin-actin complex from crude Acanthamoeba extract [23] were performed using PEGdextran two-phase systems. We have demonstrated the purification of glucosyltransferase (GTF) from Streptococcus mutans cell-lysate and S. sobrinus culture medium [26,27] using a 7.7% PEG 8000-4.0% dextran T500 solvent system. After HSCCC purification, these polymers were removed from CCC fractions using a hydroxyapatite chromatography.…”

Section: Introductionmentioning

confidence: 99%

Purification of Proteins From Cell-Culture Medium or Cell-Lysate by High-Speed Counter-Current Chromatography Using Cross-Axis Coil Planet Centrifuge

Shibusawa¹,

Ito²

2007

TOACJ

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Countercurrent chromatography as a frontier tool to discover new mechanical roles in liquid–liquid phase separation of cellular biomolecules

Shinomiya

Iijima

Ito

2023

Biomedical Chromatography

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The development of countercurrent chromatography (CCC) technology enabled us to achieve higher peak resolutions and more shortened separation times even for protein separation using aqueous two-phase solvent systems composed of polyethylene glycol and inorganic salts (or dextrans). By eliminating the solid support matrix, all analytes can be recovered from the coiled column after the separation is completed.Recently, it has been found that droplets of biomolecules formed by liquid-liquid phase separation in cells closely relate to the transcription, regulation of signal transduction, and formation of amyloids. Meanwhile, although CCC is a separation technique based on liquid-liquid partitioning of analytes between two immiscible phases, the mechanism of separation could suggest some idea concerning the formation of biomolecule droplets in cells. This article describes the recent advances in the CCC apparatus, the coiled separation column, the choice of a suitable two-phase solvent system, and the application to separation and purification of bioactive macromolecules such as proteins and enzymes, and also discusses the possibility of CCC as a tool to reveal new mechanical roles of biomolecule droplets in the cellular environments.aqueous two-phase solvent system, cellular biomolecules, coil planet centrifuge, coil satellite centrifuge, countercurrent chromatography 1 | INTRODUCTION Recently, it has been found that some proteins in cells function as aggregates after forming droplets in the liquid-liquid phase separation (LLPS) process. This discovery closely relates to various biological behaviors, including transcription, regulation of signal transduction, and formation of amyloids. The transcription factor forms droplets congregated by an electrostatic interaction between the intrinsically disordered domain and DNA and transferred into a higher state of transcription activity (Chong et al., 2018). The study on signal transduction mechanism shows that biomacromolecules connect with one another by weak interaction, and in proteins the intrinsically disordered regions generally relate to their interactions. Stress granules formed temporally in cytoplasm regulate stress response signals by holding various transduction molecules in themselves (Riggs et al., 2020). Furthermore, amyloid, protein aggregate often formed by the failure of folding, is attributed to serious diseases, that is, neurodegenerative diseases such as Alzheimer's disease, Huntington's

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Purification of Glucosyltransferase fromStreptococcus sobrinusCell Culture Medium by Combined Use of Batch Extraction and Countercurrent Chromatography with a Polymer Phase System

Cited by 4 publications

References 28 publications

Aqueous–aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins

Aqueous–aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins

Purification of Proteins From Cell-Culture Medium or Cell-Lysate by High-Speed Counter-Current Chromatography Using Cross-Axis Coil Planet Centrifuge

Countercurrent chromatography as a frontier tool to discover new mechanical roles in liquid–liquid phase separation of cellular biomolecules

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