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Method for purifying protein and glucose dehydrogenase

Inactive Publication Date: 2006-05-04
ARKRAY INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0019] Hayade has confirmed that higher enzyme activity is achieved by a combination of γ subunit and α subunit than by α subunit only. Therefore, in view of enzyme activity, it is preferable to manifest γ subunit, and when engineering the DNA, γ subunit structural gene should preferably be included in an upstream region of α subunit. Then, when the transformant produces α subunit, γ subunit which has been manifested already and existing as a protein will promote efficient production of α subunit in the microorganism.

Problems solved by technology

Shortcomings of GDH include poor thermal stability and lower substrate specificity than GOD.
However, it was not possible to purify CyGDH to a high level of purification, as SDS-PAGE examinations revealed that the obtained enzyme solution contained a number of different proteins in addition to α, β, γ subunits.

Method used

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  • Method for purifying protein and glucose dehydrogenase
  • Method for purifying protein and glucose dehydrogenase

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0064] In this Example, the enzyme solution (solubilized GDH fraction) which was obtained from KS1 strain according to the above-described technique was purified by using a combination of hydrophobic chromatography and anion exchange chromatography.

[0065] The hydrophobic chromatography was performed by using an Octyl sepharose 4 Fast Flow column (44 mm ID×20 cm Amersham Bioscience KK) which had been equilibrated with 60 mM phosphate buffer solution (pH6). The column was supplied with solubilized GDH fraction (enzyme solution) which was prepared by adding 1 M phosphate buffer solution (pH 6) to achieve the final concentration of 60 mM. Next, 600 mL of 60 mM phosphate buffer solution (pH 6) and 900 mL of 20 mM phosphate buffer solution (pH 8) were passed, and then tightly adsorbed GDH was eluted by supplying eluent. The eluent was provided by 20 mM phosphate buffer solution (pH 8) which contained sodium cholate in dissolved form. The eluent was supplied at a rate of 15 mL / min so that...

example 2

[0070] In this Example, the crude enzyme solution obtained from the transformant of Pseudomonas putida was purified by using a combination of hydrophobic chromatography and anion exchange chromatography.

[0071] In the hydrophobic chromatography, first, a Phenyl cellulofine column (300 mm ID×10 cm, Chisso Corporation, Tokyo) which had been equilibrated with 10 mM phosphate buffer solution (pH 8) containing 0.1 M KCl was supplied with the crude enzyme solution, to let the packing agent hold GDH. Next, 7 L of 10 mM phosphate buffer solution (pH 8) containing 0.1 M KCl, and 21 L of 10 mM phosphate buffer solution (pH 8) were passed. Then, tightly adsorbed GDH was eluted by passing eluent. The eluent was provided by a 20 mM phosphate buffer solution (pH 8) containing sodium cholate in dissolved form. The eluent was supplied at a rate of 7 L / min so that sodium cholate concentration would vary linearly in the range of 0-1 wt %.

[0072] As a result, GDH was eluted when the sodium cholate con...

example 3

[0077] In this Example, the crude enzyme solution obtained from the transformant of Pseudomonas putida was purified by using a combination of hydrophobic chromatography and anion exchange chromatography.

[0078] The hydrophobic chromatography was performed in the same way as in Example 2. The collected solution was ultracondensed, to yield 70 mL of phenyl collected fraction, which had a specific activity of 300 U / mg and a total activity of 21 kU.

[0079] The anion exchange chromatography was performed by using a QAE-TOYOPEARL 550 column (44 mm ID×10 cm, Tohso Corporation, Tokyo) which had been equilibrated with 10 mM phosphate buffer solution (pH8). The column was supplied with the phenyl collected fraction, and then with eluent. The eluent was provided by 10 mM phosphate buffer solution (pH8) containing 1 wt % sodium cholate. The eluent was supplied at a rate of 5 mL / min and by a volume of 2000 mL.

[0080] As a result, GDH was eluted specifically when approximately 1600 mL of the elue...

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Abstract

A method of purifying a target protein from a solution, in which the target protein containing an electron transfer protein is dissolved, with the use of liquid chromatography. The liquid chromatography is performed by introducing the above-described protein solution into a tank filled with a packing agent, thus bonding the target protein to the packing agent, removing impurities, and then eluting the target protein from the packing agent with the use of an eluent containing a hydroxycholanoic acid salt. As an example of the above protein, glucose dehydrogenase containing a protein having an activity of dehydrogenating glucose is cited. The liquid chromatography is performed by combining hydrophobic chromatography with anion exchange chromatography.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for purifying protein by using liquid chromatography. The method is used, for example, when purifying glucose dehydrogenase which is bonded to electron transfer protein. BACKGROUND ART [0002] Development efforts have been made in many different fields of industry for biosensors incorporating an enzyme which makes a specific reaction with a specific substrate. A representative example of such biosensors is a glucose sensor utilized mainly in the field of medical care. [0003] The glucose sensor establishes a reaction system which includes an enzyme and an electron transfer material. When using the glucose sensor, an amperometric method for example is employed to quantitate glucose. The enzyme is provided by e.g. glucose oxidase (GOD) and glucose dehydrogenase (GDH). [0004] GOD has high substrate specificity to glucose, high thermal stability, and is less expensive than other enzymes because it can be mass-produced indust...

Claims

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Application Information

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IPC IPC(8): C12N9/02C07K1/18C07K1/20C12N9/04
CPCC07K1/18C07K1/20C12N9/0006C12N9/0004
Inventor YAMAOKA, HIDEAKIKUROSAKA, KEISUKEKAWASE, SHIDO
Owner ARKRAY INC
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