US20060094098A1 - Method for purifying protein and glucose dehydrogenase - Google Patents

Method for purifying protein and glucose dehydrogenase Download PDF

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US20060094098A1
US20060094098A1 US10/526,026 US52602605A US2006094098A1 US 20060094098 A1 US20060094098 A1 US 20060094098A1 US 52602605 A US52602605 A US 52602605A US 2006094098 A1 US2006094098 A1 US 2006094098A1
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purifying
protein
glucose dehydrogenase
cholate
eluent
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Hideaki Yamaoka
Keisuke Kurosaka
Shido Kawase
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Arkray Inc
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Arkray Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)

Definitions

  • 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.
  • 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.
  • the glucose sensor establishes a reaction system which includes an enzyme and an electron transfer material.
  • an amperometric method for example is employed to quantitate glucose.
  • the enzyme is provided by e.g. glucose oxidase (GOD) and glucose dehydrogenase (GDH).
  • GOD has high substrate specificity to glucose, high thermal stability, and is less expensive than other enzymes because it can be mass-produced industrially.
  • a shortcoming on the other hand is that reaction systems involving GOD are highly sensitive to oxygen dissolved in the sample, and so the dissolved oxygen can affect the measurement.
  • reaction systems involving GDH are not susceptible to dissolved oxygen in the sample. For this reason, reaction systems which utilize GDH allow accurate measurement of the glucose level even if the measurement is made in an environment poor in oxygen partial pressure, or if the measurement is made to a high-concentration sample which has a high oxygen demand. Shortcomings of GDH include poor thermal stability and lower substrate specificity than GOD.
  • CyGDH When utilizing CyGDH in glucose sensors, CyGDH must be purified from an enzyme solution which contains CyGDH. Enzyme is usually purified by means of liquid chromatography, so the inventor et al. of the present invention followed a common method of hydrophobic chromatography and anion exchange chromatography in an attempt to process the enzyme solution. 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 ⁇ , ⁇ , ⁇ subunits.
  • the present invention aims at providing a new method for purifying a protein.
  • a first aspect of the present invention provides a method for purifying a target protein from a protein solution which contains the target protein, by using liquid chromatography.
  • the liquid chromatography includes: a first step of introducing the protein solution into a column which is filled with a packing agent and causing the packing agent to hold the target protein; and a second step of eluting the target protein by using an eluent which contains a hydroxy-cholate.
  • liquid chromatography refers, unless otherwise specified, to both of a column method in which the purification is made in a flow (continuously), and a batch method of purifying protein.
  • An example of the batch method is placing a packing agent and the protein solution together in a column to cause the packing agent to bind the target protein, then separating the packing agent, and then bringing the packing agent in contact with the eluent thereby separating and collecting the target agent from the packing agent.
  • target protein which is the object of the purification
  • target protein is a protein which contains electron transfer protein.
  • target protein is glucose dehydrogenase which contains an electron transfer protein and a protein that has glucose dehydrogenation activity.
  • the packing agent can be an ion-exchange gel, and the protein is purified by means of an ion-exchange chromatography.
  • the ion-exchange gel contains a quaternary ammonium group as an ion-exchange group.
  • hydroxy-cholate refers to salts of cholic acid which are trihydrates of cholanic acids, as well as their derivatives in a broad sense.
  • hydroxy-cholates include cholates, glicoursodeoxycholate, tauroglicoursodeoxycholate, tauroursodeoxycholate, ursodeoxycholate, glycocholate, taurocholate, glycochenodeoxycholate, taurochenodeoxycholate, glycodeoxycholate, taurodeoxycholate, chenodeoxycholate, deoxycholate, glycolithocholate, taurolithocholate, and lithocholate.
  • a cholate such as sodium cholate.
  • the concentration of the hydroxy-cholate in the eluent is maintained at a constant level.
  • the concentration of the hydroxy-cholate in the eluent is preferably selected from a range of 0.5 through 2.5 wt %, and more preferably, from 0.8 through 1.2 wt %.
  • concentration of the hydroxy-cholate in the eluent may be varied with time, in eluting the target protein.
  • the upper limit concentration of the eluting agent should be not higher than 3 wt % for example, and more preferably not higher than 1.5 wt %, and most preferably not higher than 1 wt %.
  • the concentration of the eluent is varied within the range of 0-3 wt % for example, more preferably within the range of 0-1.5 wt %, and most preferably within the range of 0-1.0 wt %.
  • the electron transfer protein has a molecule weight of e.g. approximately 43 kDa in SDS-gel electrophoresis under a reducing environment.
  • the protein which has glucose dehydrogenation activity has a molecule weight of approximately 60 kDa in SDS-gel electrophoresis under a reducing environment.
  • Glucose dehydrogenase which contains such electron transfer protein and subunits is obtainable from e.g. a microorganism belonging to the genus Burkholderia which is capable of producing the glucose dehydrogenase, or from a transformant thereof.
  • KS1 strain Burkholderia cepacia , and Burkholderia cepacia KS1 strain (hereinafter simply called “KS1 strain”) in particular, is preferred.
  • KS1 strain was deposited as FERM BP-7306, on Sep. 25, 2000, with the International Patent Organism Depositary (IPOD), National Institute of Advanced Industrial Science and Technology (AIST) (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566).
  • the transformant can be produced, for example, by engineering a host microorganism with a DNA from a microorganism belonging to the genus Burkholderia for coding the electron transfer protein and the protein active against glucose.
  • the host microbe is preferably a microorganism belonging to the genus Pseudomonas ( Pseudomonas putida in particular) or E. coli bacterium.
  • glucose dehydrogenase derived from KS1 strain contains a subunit ( ⁇ subunit) and an electron transfer protein ( ⁇ subunit), and ⁇ subunit which has a molecular weight of approximately 14 kDa in the SDS gel-electrophoretic migration under reducing environment.
  • Hayade has confirmed that higher enzyme activity is achieved by a combination of ⁇ subunit and ⁇ subunit than by ⁇ subunit only. Therefore, in view of enzyme activity, it is preferable to manifest ⁇ subunit, and when engineering the DNA, ⁇ subunit structural gene should preferably be included in an upstream region of ⁇ subunit. Then, when the transformant produces ⁇ subunit, ⁇ subunit which has been manifested already and existing as a protein will promote efficient production of ⁇ subunit in the microorganism.
  • a second aspect of the present invention provides a method for purifying glucose dehydrogenase using a combination of hydrophobic chromatography and anion exchange chromatography.
  • the hydrophobic chromatography includes: a step of causing a stationary phase to hold the glucose dehydrogenase; a step of eluting unnecessary proteins; and a step of eluting the glucose dehydrogenase by using an eluent containing a hydroxy-cholate.
  • the anion exchange chromatography includes: a step of causing a stationary phase to hold the glucose dehydrogenase; and a step of eluting the glucose dehydrogenase by using an eluent containing a hydroxy-cholate.
  • elution of the glucose dehydrogenase is performed by varying the concentration of the hydroxy-cholate in the eluent with time.
  • elution of the glucose dehydrogenase is performed by keeping the concentration of the hydroxy-cholate in the eluent.
  • the concentration of the hydroxy-cholate in the eluent may be varied with time, in eluting the target protein.
  • the upper limit concentration of the eluting agent should be not higher than 3 wt % for example, and more preferably not higher than 1.5 wt %, and most preferably not higher than 1 wt %.
  • the concentration of the eluent is varied within the range of 0-3 wt % for example, more preferably within the range of 0-1.5 wt %, and most preferably within the range of 0-1.0 wt %.
  • the anion exchange chromatography is carried out after the hydrophobic chromatography.
  • the glucose dehydrogenase is produced by e.g. a microorganism belonging to the genus Burkholderia .
  • An example of the microorganism belonging to the genus Burkholderia is KS1 strain.
  • the glucose dehydrogenase may be made by a transformant.
  • the transformant can be produced by engineering a host microorganism with a DNA from a microorganism belonging to the genus Burkholderia for coding the glucose dehydrogenase.
  • the host microbe can be Pseudomonas putida or E. coli bacterium for example.
  • the anion exchange chromatography is performed by using an ion-exchange gel which contains a quaternary ammonium group as an ion-exchange group, and the hydroxy-cholate is provided by a cholate.
  • FIG. 1 shows an SDS-PAGE result of an enzyme which was purified with a sodium cholate eluent from a crude enzyme solution obtained from a transformant of Pseudomonas putida.
  • FIG. 2 shows an SDS-PAGE result of an enzyme which was purified with a NaCl or KCl eluent from a crude enzyme solution obtained from the transformant of Pseudomonas putida.
  • FIG. 3 shows an SDS-PAGE result of an enzyme which was purified with a sodium cholate eluent from a crude enzyme solution obtained from a transformant of E. coli bacterium, together with results of two enzymes; one of which was purified from a crude enzyme solution obtained from a transformant of Pseudomonas putida while the other was purified from a crude enzyme solution obtained from KS1 strain.
  • GDH glucose dehydrogenase
  • an enzyme solution is prepared.
  • the enzyme solution may be obtained from a microbe which produces the glucose dehydrogenase, a culture of the microbe, a transformant derived through insertion of DNA which is extracted from the microbe, or a culture of the transformant.
  • the enzyme solution can be obtained by first separating the microbe from the culture through filtration, centrifugation, etc., and then fragmentizing the microbe mechanically or enzymatically with e.g. lysozyme, followed as necessary, by solubilization of GDH through addition of a chelating agent such as EDTA and a surfactant.
  • a chelating agent such as EDTA and a surfactant.
  • the enzyme solution can be obtained by separating the microbe from the culture through filtration, centrifugation and so on.
  • microbes which produce glucose dehydrogenase microbes of the genus Burkholderia , and Burkholderia cepacia in particular is preferably utilized.
  • Burkholderia cepacia it is possible to obtain the enzyme solution in the form of a solubilized membrane fraction for example.
  • the solubilized membrane fraction for example, first the microbe is centrifugally separated from a culture, and the microbe is fragmentized to obtain a cell extract. The cell extract is then centrifuged, and thereafter, obtained supernatant fluid is super-centrifuged. The resulting sediment is the solubilized membrane fraction.
  • the fragmentization of the microbe can be made in an ordinary mechanical or enzymatic method.
  • the transformant can be produced by first obtaining DNA which codes manifestation of e.g. ⁇ subunit (a protein having glucose dehydrogenating activity) and ⁇ subunit (electron transfer protein), and then introducing the DNA into a host microbe by using a recombinant vector.
  • ⁇ subunit a protein having glucose dehydrogenating activity
  • ⁇ subunit electron transfer protein
  • a recombinant vector is constructed.
  • the construction of recombinant vector can be achieved by first separating and purifying a chromosome DNA from a microbe which produces an enzyme having glucose dehydrogenation activity, preparing the chromosome DNA fragments through shredding or PCR amplification, and then binding and closing the chromosome DNA fragments with a linear expression vector.
  • Examples of the host microbe include E. coli and other enteric bacteria, gram-negative bacteria such as the genus pseudomonas and the genus Gluconobacter , gram-positive bacteria including the genus Bacillus such as Bacillus subtilis , yeasts such as Saccharomyces cerevisiae , and filamentous bacteria such as Aspergillus niger .
  • E. coli bacteria and those of the genus pseudomonas pseudomonas putida for example
  • the transformation of the microbes can be made by competent cell method through calcium treatment for the genus Escherichia .
  • Protoplast methods can be used for the genus bacillus , KU method and KUR method for yeasts, and micromanipulation methods for filamentous bacteria. Transformation can also be made by using electropolation method.
  • the enzyme solution is purified by using liquid chromatography.
  • liquid chromatography In performing the purification through liquid chromatography, a selection is made for the type, number and combination of different types of chromatography so that a target level of purification can be achieved.
  • Usable types of liquid chromatography include gel filtration chromatography, adsorption chromatography, ion exchange chromatography and affinity chromatography.
  • the liquid chromatography may be made by first having the target protein captured in the stationary phase in the column, and then supplying eluent continuously thereby eluting the target protein.
  • a batch method may be used. In the batch method, for example, a column is supplied with a packing agent and a solution which contains a target protein so the packing agent will hold the target protein. Then, impurities are removed, and eluent is supplied to elute the target protein from the packing agent for collection.
  • the eluent is a solution which contains a hydroxy-cholate as an eluting agent. If the liquid chromatography is performed for a plurality of times before yielding the purified target protein, hydroxy-cholate is used as the eluent at least in one cycle of the liquid chromatography. In this case, preferably, hydroxy-cholate is used in the last cycle of liquid chromatography, as the eluent.
  • hydroxy-cholate examples include cholates, glicoursodeoxycholate, tauroglicoursodeoxycholate, tauroursodeoxycholate, ursodeoxycholate, glycocholate, taurocholate, glycochenodeoxycholate, taurochenodeoxycholate, glycodeoxycholate, taurodeoxycholate, chenodeoxycholate, deoxycholate, glycolithocholate, taurolithocholate and lithocholate.
  • cholate such as sodium cholate is preferred.
  • the concentration of the hydroxy-cholate in the solvent may be kept at a constant level, or the hydroxy-cholate concentration may be linearly changed with the time during the supply.
  • the liquid chromatography may be made directly to the enzyme solution, or may be made after the target protein in the enzyme solution has been concentrated.
  • concentration can be made by e.g. vacuum concentration, membrane concentration, salting-out procedure, factional precipitation using hydrophilic solvent (e.g. methanol, ethanol and acetone), heating, and isoelectric process.
  • hydrophilic solvent e.g. methanol, ethanol and acetone
  • the purified enzyme thus obtained is made into a powdery product through such a process as freeze-drying, vacuum-drying and spray-drying, for distribution in the market.
  • Burkholderia cepacia KS1 strain was cultured under an aerobic condition. More specifically, the KS1 strain was cultured in 20 L of medium at 34° C. for 8 hours. The medium contained ingredients listed in Table 1, per liter. TABLE 1 MEDIUM COMPOSITION Polypeptone 10 g Yeast extract 1 g NaCl 5 g KH 2 PO 4 2 g Glucose 5 g Einol (ABLE Co., Tokyo Japan) 0.14 g Total, distilled water 1 L pH 7.2
  • the 20 L of culture medium was centrifuged at 4° C., for 10 minutes at 9000 ⁇ g, to obtain approximately 250 g of microbe body.
  • the collected microbe body was frozen, then suspended in 10 mM phosphate buffer solution (pH6), and processed in a high-pressure homogenizer (manufactured by Rannie, Denmark) several times at a pressure of 500 bar, to fragmentize cell membrane.
  • the cell extract obtained by cell membrane fragmentation showed a GDH activity of 60 kU.
  • the cell extract was then centrifuged under 8000 ⁇ g for 60 minutes, to remove cell solid. Further, the supernatant liquid was super-centrifuged at 10° C. under 170,000 ⁇ g for an hour, to collect membrane fraction as the sediment.
  • the membrane fraction was dissipated in 10 mM phosphate buffer solution (pH 6), so as to have a final sodium cholate concentration of 1.5%, and KCl concentration of 0.1 M, and steered at 4° C. over a night. As a result, membrane fraction suspension which contained GDH at 30 kU was obtained.
  • the membrane fraction suspension was super-centrifuged at 10° C. under 170,000 ⁇ g for 90 minutes, to remove sediment, and to obtain GDH-containing solubilized membrane fraction (at GDH activity of 26 kU).
  • the solubilized membrane fraction was dialyzed with 10 mM phosphate buffer solution (pH 6) for 3 nights, and the obtained insoluble matter was removed as sediment in a super centrifugation at 10° C. under 170,000 ⁇ g for 90 minutes.
  • the obtained supernatant liquid (solubilized GDH fraction) contained GDH which showed an activity of 28 kU and a specific activity of 6.8 U/mg.
  • DNA which includes sequences for coding manifestation of ⁇ , ⁇ and ⁇ subunits was obtained from KS1 strain according to a common method.
  • the obtained DNA was inserted into a vector plasmid, to produce a GDH expression plasmid, which was introduced into host microbes, to make transformants.
  • the hosts were provided by Pseudomonas putida KT2440 strain (ATCC 47054) and E. coli bacterium BL21 strain.
  • Pseudomonas putida transformant was cultured under normal aerobic conditions, in 20 L of culture solution.
  • Composition of the culture solution included 3.2% polypeptone, 2% yeast extract, 0.5% NaCl, 2% glycerol, 0.05 mL/L Adekanol LG-126 (Asahi Denka Co., Ltd., Tokyo) and 50 ⁇ g/mL streptomycin (pH 7.0).
  • 200 mL of the previous culture solution was inoculated and culture was started at 34° C.
  • IPTG Isopropyl- ⁇ - D -thiogalactopyranoside
  • culturing was continued further for 20 hours, to obtain the target culture solution.
  • This culture solution was centrifuged by a Sharpless centrifuge, and approximately 800 g of Pseudomonas putida transformant was obtained.
  • E. coli bacterium transformant was also cultured under normal aerobic conditions, in 2 L of culture solution.
  • Composition of the culture solution included 3.2% polypeptone, 2% yeast extract, 0.5% NaCl, 2% glycerol, 0.05 mL/L Adekanol LG-126 (Asahi Denka Co., Ltd., Tokyo), 50 ⁇ g/mL ampicillin, and 50 ⁇ g/mL kanamycin (pH 7.0).
  • 50 mL of the previous culture solution was inoculated and cultured at 30° C. for 29 hours. The culture solution was centrifuged, and approximately 85 g of E. coli bacterium transformant was obtained.
  • microbe transformed
  • 10 mM phosphate buffer solution pH 8
  • Mydol 12 Kao Corporation, Tokyo
  • KCl KCl
  • Pseudomonas putida transformant was found to have a GDH activity of 2930 kU, and a specific activity of 22 U/mg.
  • E. coli bacterium transformant was found to have a GDH activity of 259 kU, and a specific activity of 10.3 U/mg.
  • Glucose dehydrogenation activity was measured by tracking a redox reaction of electron acceptor based on glucose dehydrogenation.
  • the electron acceptors were provided by 2,6-dichlorophenol-indophenol (DCIP) and phenazine methosulfate (PMS).
  • 900 ⁇ L of 47 mM phosphate buffer solution (pH 6.0) containing 20 mM glucose, 2 mM PMS and 0.1 mM DCIP was placed in a spectrophotometer cell, and was pre-incubated at 37° C. for 3 minutes. Next, 0.5-10 ⁇ L of the enzyme solution was added. The cell was immediately turned upside down to begin reaction, and time course monitoring was made for absorption drop at a 600 nm wavelength at 37° C. DCIP has an absorption wavelength of 600 nm and the absorption drop is due to a redox reaction of the electron acceptors based on glucose dehydrogenation.
  • 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.
  • 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.
  • solubilized GDH fraction enzyme solution
  • 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 sodium cholate concentration would vary linearly in the range of 0-1 wt %.
  • GDH was eluted when the sodium cholate concentration was approximately 0.8 wt %, yielding 340 mL of GDH active fraction.
  • the collected fraction (Octyl collected fraction) had activity readings of 108 U/mg specific activity and 16 kU of total activity.
  • the anion exchange chromatography was performed by using a Q sepharose Fast Flow column (32 mm ID ⁇ 12 cm Amersham Bioscience KK) which had been equilibrated with 20 mM phosphate buffer solution (pH 8). To this column, the Octyl collected fraction was supplied, and then eluent was supplied. The eluent was provided by 20 mM phosphate buffer solution (pH 8) which contained 1 wt % sodium cholate. The eluent was supplied at a rate of 8 mL/min and by a volume of 600 mL.
  • GDH was eluted specifically when approximately 300 mL of the eluent had been passed, yielding 340 mL of a GDH active fraction.
  • the collected fraction (Q collected fraction) had 770 U/mg specific activity, and 14 kU total activity.
  • the crude enzyme solution obtained from the transformant of Pseudomonas putida was purified by using a combination of hydrophobic chromatography and anion exchange chromatography.
  • 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 %.
  • the anion exchange chromatography was performed by using a Q sepharose Fast Flow column (44 mm ID ⁇ 20 cm Amersham Bioscience KK) which had been equilibrated with 10 mM phosphate buffer solution (pH8).
  • the column was supplied with the Phenyl collected fraction. Note that the phenyl collected fraction was buffer-substituted to 10 mM phosphate buffer solution (pH 8) by using a laboratory module (Asahi Kasei Corporation, Tokyo) which had a 50000 molecular weight cutoff, before being supplied to the column.
  • the column was supplied with 600 mL of 10 mM phosphate buffer solution (pH 8), and then with eluent.
  • the eluent was provided by 10 mM phosphate buffer solution (pH 8) containing 1 wt % sodium cholate. The eluent was supplied at a rate of 10 mL/min.
  • GDH was eluted specifically when approximately 1400 mL of the eluent had been passed, yielding 313 mL of a GDH active fraction.
  • the collected fraction (Q collected fraction) had 1283 U/mg specific activity, and 390 kU total activity.
  • the phenyl collected fraction and the Q collected fraction were subjected to electrophoresis, i.e. SDS-PAGE.
  • the SDS-PAGE used Tris-Tricine buffer solution and was performed in polyacrylamide buffer solution in 8-25% gel gradient. Protein which migrated in the gel was stained with CBB. Results of the SDS-PAGE are shown in FIG. 1 .
  • Lane 2 represents CBB-stained Phenyl collected fraction and Lane 3 represents CBB-stained Q collected fraction.
  • the crude enzyme solution obtained from the transformant of Pseudomonas putida was purified by using a combination of hydrophobic chromatography and anion exchange chromatography.
  • 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.
  • 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.
  • GDH was eluted specifically when approximately 1600 mL of the eluent has been passed, yielding 300 mL of active fraction.
  • the collected fraction (QAE collected fraction) had 1500 U/mg specific activity, and 7.8 kU total activity.
  • the QAE collected fraction was subjected to SDS-PAGE, using the same procedure as used in Example 2, and the protein was stained with CBB. Results of the SDS-PAGE are shown in FIG. 1 .
  • Lane 4 represents CBB-stained QAE collected fraction.
  • the hydrophobic chromatography was performed in the same way as in Example 2, yielding 7200 mL of phenyl collected fraction, which had specific activity of 314 U/mg and total activity of 256 kU. In this Comparative Example, however, a portion of the phenyl collected fraction or 1100 mL (Q apply) which represents a total activity of 39 kU was subjected to the following anion exchange chromatography.
  • the anion exchange chromatography was performed by using a Q sepharose Fast Flow column (44 mm ID ⁇ 13 cm Amersham Bioscience KK) which had been equilibrated with 10 mM phosphate buffer solution (pH 8).
  • the column was supplied with the Q apply. Thereafter, the column was supplied with 800 mL of 10 mM phosphate buffer solution (pH 8) in order to remove non-adsorbent protein. Then, the column was supplied with eluent.
  • the eluent was provided by 10 mM phosphate buffer solution (pH 8) which contained NaCl in dissolved form.
  • the eluent was supplied at a rate of 7.5 L/min so that NaCl concentration would vary linearly in the range of 0-0.6 M.
  • GDH was eluted at two NaCl concentration levels of 0.25 M approx. and 0.4 M approx., yielding 140 mL and 360 mL of active fractions respectively.
  • Each of the active fractions (Q collected fraction (1) and Q collected fraction (2)) were subjected to activity measurement.
  • the Q collected fraction (1) had specific activity of 600 U/mg and total activity of 4.5 kU.
  • the Q collected fraction (2) had specific activity of 432 U/mg and total activity of 12 kU.
  • the phenyl collected fraction and the Q collected fraction (2) were subjected to SDS-PAGE using the same procedure as used in Example 2, and the protein was stained with CBB. Results of the SDS-PAGE are shown in FIG. 2 .
  • Lane 2 represents the phenyl collected fraction
  • Lane 3 represents the Q collected fraction (1).
  • 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 (pH 8).
  • the column was first supplied with the QAE collected fraction (74 kU total activity).
  • 800 mL of 10 mM phosphate buffer solution (pH 8) was supplied to remove non-adsorbent protein.
  • the column was supplied with eluent.
  • the eluent was provided by 10 mM phosphate buffer solution (pH 8) containing dissolved KCl.
  • the eluent was supplied at a rate of 5 mL/min so that KCl concentration would vary linearly in the range of 0-1M.
  • GDH was eluted at two KCl concentration levels of 0.23 M approx. and 0.43 M approx., yielding 200 mL and 400 mL of active fractions respectively.
  • Each of the active fractions (QAE collected fraction (1) and QAE collected fraction (2)) were subjected to activity measurement.
  • the QAE collected fraction (1) had specific activity of 399 U/mg and total activity of 7.5 kU.
  • the QAE collected fraction (2) had specific activity of 217 U/mg and total activity of 6.4 kU.
  • the QAE collected fraction was subjected to SDS-PAGE using the same procedure as used in Example 2, and the protein was stained with CBB. Results of the SDS-PAGE are shown in FIG. 2 .
  • Lane 4 represents the QAE collected fraction.
  • the crude enzyme solution obtained from the transformant of E. coli bacterium was purified by using a combination of hydrophobic chromatography and anion exchange chromatography.
  • the hydrophobic chromatography was performed under the same conditions as in Example 1, using an Octyl sepharose 4 Fast Flow column.
  • GDH was eluted at the sodium cholate concentration of approximately 0.8 wt %, yielding 220 mL of GDH active fraction.
  • the collected fraction (Octyl collected fraction) had specific activity of 503 U/mg and total activity of 149 kU.
  • the anion exchange chromatography was performed by using a Q sepharose Fast Flow column and under the same conditions as in Example 1.
  • GDH was eluted specifically when approximately 500 mL of the eluent had been passed, yielding 175 mL of GDH active fraction.
  • the collected fraction (Q collected fraction) had 1147 U/mg specific activity, and 50 kU total activity.
  • the Q collected fraction was subjected to SDS-PAGE using the same procedure as used in Example 2, and the protein was stained with CBB. Results of the SDS-PAGE are shown in FIG. 3 .
  • Lane 3 represents the Q collected fraction according to the present Example.
  • the Q collected fraction (Lane 2) according to Example 1 and the Q collected fraction (Lane 4) according to Example 2 are also subjected to the electrophoresis.
  • GDH has a higher final specific activity and is purified more efficiently when the eluent is provided by cholate (Example 1 through 4) than when the eluent is provided by NaCl or KCl and GDH is purified through gradient elution (Comparative Example 1 and 2). This is also indicated in FIG. 1 and FIG. 2 .
  • GDH derived from the KS1 strain includes ⁇ , ⁇ and ⁇ subunits, and their molecular weights in the SDS gel-electrophoretic migration under reducing environment were approximately 60 kDa, 43 kDa and 14 kDa respectively.
  • Example 2 will reveal that the collected fractions in Example 2 and Example 3 have wider bands which represent ⁇ , ⁇ and ⁇ subunits, than the corresponding bands found in the collected fractions in Comparative Examples 1 and 2, while having smaller portions of proteins which have different molecular weights. Therefore, when purifying GDH by using a combination of hydrophobic chromatography and anion exchange chromatography, use of sodium cholate for eluting GDH will lead to efficient purification of GDH.
  • crude enzyme derived from a transformant of host E. coli bacterium shows a narrower band for the ⁇ subunit but wider bands for ⁇ and ⁇ subunits than other crude enzyme solutions.
  • E. coli bacterium is advantageous in that it is inexpensive, easily available and has superb self-duplicating capability. Therefore, in terms of industrial application, the method of obtaining a crude enzyme from a transformant hosted by E. coli bacterium and purifying this crude enzyme to obtain GDH will be useful.

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US20060258959A1 (en) * 2003-09-02 2006-11-16 Koji Sode Glucose sensor and glucose level measuring apparatus
TWI400128B (zh) * 2009-11-30 2013-07-01 Univ Tamkang 蛋白質純化方法
US9488625B2 (en) 2010-12-15 2016-11-08 Baxalta GmbH Purification of factor VIII using a conductivity gradient

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USRE45764E1 (en) 2001-09-14 2015-10-20 Arkray, Inc. Concentration measuring method, concentration test instrument, and concentration measuring apparatus
DE602005025104D1 (de) 2004-04-23 2011-01-13 Arkray Inc Mutierte glucosedehydrogenase
JP5598810B2 (ja) * 2009-04-10 2014-10-01 国立大学法人三重大学 コイ由来抗菌剤の製造方法及び抗菌処理装置
TW201221641A (en) * 2010-10-11 2012-06-01 Abbott Lab Processes for purification of proteins

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US5939536A (en) * 1990-03-05 1999-08-17 Genzyme Corporation Methods for purifying cystic fibrosis transmembrane conductance regulation
US20040023330A1 (en) * 2000-10-31 2004-02-05 Koji Sode Novel glucose dehydrogenase and process for producing the dehydrogenase

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DE3711881A1 (de) * 1987-04-08 1988-10-27 Merck Patent Gmbh Verfahren zur herstellung von glucosedehydrogenase aus bacillus megaterium

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US5250415A (en) * 1987-04-08 1993-10-05 Merck Patent Gesellschaft Mit Beschrankter Haftung Process for the preparation of glucose dehydrogenase from Bacillus megaterium
US5939536A (en) * 1990-03-05 1999-08-17 Genzyme Corporation Methods for purifying cystic fibrosis transmembrane conductance regulation
US20040023330A1 (en) * 2000-10-31 2004-02-05 Koji Sode Novel glucose dehydrogenase and process for producing the dehydrogenase

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060258959A1 (en) * 2003-09-02 2006-11-16 Koji Sode Glucose sensor and glucose level measuring apparatus
US7497940B2 (en) * 2003-09-02 2009-03-03 Koji Sode Glucose sensor and glucose level measuring apparatus
US20090177067A1 (en) * 2003-09-02 2009-07-09 Arkray, Inc. Glucose Sensor and Glucose Level Measuring Apparatus
US8277636B2 (en) 2003-09-02 2012-10-02 Koji Sode Glucose sensor and glucose level measuring apparatus
TWI400128B (zh) * 2009-11-30 2013-07-01 Univ Tamkang 蛋白質純化方法
US9488625B2 (en) 2010-12-15 2016-11-08 Baxalta GmbH Purification of factor VIII using a conductivity gradient

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