JPH04258287A - Production of luciferase of cypridina hilgendorfi - Google Patents

Abstract

PURPOSE:To industrially produce high-purity luciferase of Cypridina hilgendorfi. CONSTITUTION:A culture solution of gene recombinant obtained by integrating luciferase of Cypridina hilgendorfi into a gene is purified by one or more kinds of chromatographies elected from ion exchange chromatography, hydrophobic chromatography and affinity chromatography.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention is directed to the marine life Cypridina (C.
The present invention relates to a method for producing luciferase of ypridina hilgendorfi from a culture solution of a recombinant that has integrated the gene.

0002

[Prior Art] In recent years, the DNA probe method has attracted attention as a new diagnostic method with high specificity for elucidating the cause of disease at the genetic level. However, since radioactive isotopes are used in the detection method, It has not become widely available due to restrictions such as personnel, facilities, and radioactive waste. Therefore, for the spread of DNA probe methods, it is necessary to develop methods that do not use radioactive isotopes, but detection systems such as colorimetric methods and fluorescence methods have low sensitivity and are not practical, so new detection systems are needed to replace them. development is desired.

[0003] Currently, research is being conducted focusing on the bioluminescence method, which has a detection sensitivity comparable to that of using radioisotopes.Up until now, luciferase from fireflies and luminescent bacteria has been used for bioluminescence. Although,
Because the enzyme itself is unstable, the expected sensitivity has not been achieved. However, Cypridina luciferase has good stability and strong luminescence intensity, and is expected to be applied to DNA probe methods, enzyme immunoassays, etc. as a highly sensitive detection system using bioluminescence.

[0004] Sea fireflies are two to three species that live along the coast of Japan.
This is a marine crustacean that lives in the sand on the ocean floor during the day, swims at night in search of food such as dead fish, and releases luciferin and luciferase into the seawater when stimulated. In seawater, luciferin is oxidized by the enzyme luciferase and dissolved oxygen in seawater, and it emits light [Goto et al.: Pharmacia, 3
, 125 (1967)]. This reaction is a very simple luminescent system that does not require other components such as ATP for luminescence, as in fireflies and luminescent bacteria [Biolumine
scene in progress, 11
5p. (1966) Princeton Univ.
Press].

[0005] As a method for isolating luciferase from Cypridina, Cypridina is freeze-dried and pulverized.
After extraction with a buffer solution, solvent precipitation and ammonium sulfate precipitation were performed.
After dialysis, purification by weak ion exchange chromatography and electrophoresis [F. I. Tsuji et al., Method
s Enzymol. , 57, 364-372 (19
78)] has been carried out. However, in order to industrially produce luciferase from natural sea fireflies, a large amount of sea fireflies is required, but the number of sea fireflies is decreasing due to ocean pollution, etc., and it is extremely difficult to secure the raw material.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide an industrial method for producing highly purified Cypridina luciferase.

[0007]

[Means for Solving the Problems] As a result of intensive research into an industrial method for producing Cypridina luciferase, the present inventors have found that the luciferase secreted into the culture solution of a recombinant that has incorporated the Cypridina luciferase gene. , they discovered that they can be purified to a high degree of purity by one or a combination of chromatography methods, and completed the present invention. That is, the present invention provides a Cypridina luciferase gene, which is characterized in that a culture solution of a genetically recombinant Cypridina luciferase gene is purified by one or more of ion-exchange chromatography, hydrophobic chromatography, and affinity chromatography. This is a method for producing luciferase.

[0008] The genetically recombinant plant incorporating the Cypridina luciferase gene of the present invention is, for example, WO90/0
It can be produced by the method described in No. 1542. That is, it can be produced by transforming cells with a recombinant vector obtained by ligating the Cypridina luciferase gene to a vector that can be expressed in various host cells. The host is not particularly limited, but yeast is preferably used. Next, culture the recombinant,
Highly purified Cypridina luciferase can be produced by removing the recombinant from the culture solution by, for example, centrifugation and purifying the supernatant using the chromatography of the present invention.

In the ion exchange chromatography of the present invention, an ion exchanger in which various functional groups are introduced into polysaccharides such as cellulose, agarose, and dextran, or synthetic polymers such as polyvinyl is used, but the isoelectric point of luciferase is Among these ion exchangers, a weakly basic anion exchanger such as diethylaminoethyl (DEAE) ion exchanger is preferably used because it has a pH of 4 to 5 and is unstable at a pH of 6 or less. Adsorption of luciferase is carried out by desalting by dialysis or gel filtration to lower the ionic strength, preferably at pH 7 to 8. Elution from the ion exchanger is carried out by increasing the ionic strength stepwise or with a gradient, preferably at pH 7-8.

In the hydrophobic chromatography of the present invention, a carrier is used in which a hydrophobic group such as a phenyl group or a butyl group is introduced as a ligand into a polysaccharide such as cellulose, agarose, or dextran, or a synthetic polymer such as polyvinyl. Adsorption of luciferase is carried out under conditions where hydrophobic bonds are likely to occur, for example, by adding 1 to 3 M ammonium sulfate to increase the ionic strength. For elution from a hydrophobic carrier, a method is preferably used in which the salt concentration is lowered stepwise or with a gradient. All of the above operations are preferably carried out at a pH of 7 to 8.

[0011] In the affinity chromatography of the present invention, a carrier is used in which a ligand is bound to a polysaccharide such as cellulose, agarose, or dextran, or a synthetic polymer such as polyvinyl through a functional group. Among these, heparin affinity carriers or metal chelate affinity carriers are preferably used. The metal to be bound to the metal chelate affinity carrier is:
Preferably copper ions are used. The adsorption conditions for the heparin affinity carrier are preferably desalting by dialysis or gel filtration to lower the ionic strength, while elution is preferably performed by increasing the ionic strength stepwise or with a gradient. . All of the above operations
It is preferable to conduct the reaction at a pH of 7 to 8. The conditions for adsorption to the copper chelate affinity carrier are to lower the ionic strength by desalting by dialysis or gel filtration at pH 7 to 8, while elution is performed by increasing the ionic strength stepwise or with a gradient. method is preferred.

[0012] The chromatography of the present invention may be used alone or in combination of two or more. Preferred examples of the combinations will be described below, but the combinations are not limited thereto. The culture solution from which the recombinant plant incorporating the Cypridina luciferase gene has been removed is adjusted to pH
The luciferase is adsorbed onto a weakly basic anion exchange carrier equilibrated with a buffer having a pH of 5 to 10, preferably 7 to 8, and the ionic strength of the buffer is increased to elute the luciferase. Ammonium sulfate is added to the eluted active fraction to an extent that luciferase does not precipitate, preferably at a saturation concentration of 30 to 40%, and this is mixed with
The luciferase is adsorbed onto a hydrophobic carrier equilibrated with a buffer containing 3M ammonium sulfate at pH 5 to 10, preferably pH 7 to 8, and the ammonium sulfate concentration of the buffer is lowered to elute the luciferase. Next, the active fraction is desalted using dialysis or a gel filtration carrier,
Highly purified recombinant Cypridina luciferase is obtained by performing chromatography using a metal chelate affinity carrier or a heparin affinity carrier.

The luciferase activity is measured by using synthetic Cypridina luciferin as a substrate and measuring the luminescence produced by luciferase with a luminometer. Displays the amount of luminescence measured with a luminometer in cps,
Let it be luciferase activity. The recombinant Cypridina luciferase obtained by this production method is
Single band in electrophoresis, specific activity 1x1013c
ps/mg protein, which is a very high purity product. In addition, the molecular weight in SDS electrophoresis under reduced conditions is approximately 60,
Indicates 000.

[0014]

[Examples] Examples of the present invention will be shown below, but the present invention is not limited thereto. Example 1 30 L of a yeast recombinant (S. cerevisiae pGL1) incorporating Cypridina luciferase gene prepared according to the method described in WO90/01542
Cultivate in a culture tank, remove bacterial cells, and collect 24L of supernatant liquid.
Obtained. Luciferase activity is 2.9x1012 in total
cps (specific activity: 1.5x109 cps/mg・pr
It was (Otein).

[0015] This supernatant was adsorbed onto DEAE/Cellulofine A500 (carrier: 1,000 ml, manufactured by Seikagaku Corporation) which had been equilibrated in advance with 20 mM Tris-HCl buffer (pH 7.1). Wash the column with equilibration buffer and
Luciferase was eluted with a linear gradient of NaCl from M to 0.3M. 1.6x1012cps (specific activity: 2.2x1010cps/mg protein)
of luciferase was recovered, and the activity recovery rate was 55%.
Met.

Butyl Toyopearl 65 was prepared by adding 30% saturated ammonium sulfate to 600 ml of the active fraction and equilibrating it with 20 mM Tris-HCl buffer (pH 7.1) containing 1M ammonium sulfate.
It was adsorbed onto 0M (carrier: 200 ml, manufactured by Tosoh Corporation). The column was washed with the same buffer as for equilibration (containing 1M ammonium sulfate), and the luciferase was eluted with a linear ammonium sulfate gradient from 1M to 0M. 1.3x1012cps (specific activity:
Luciferase (1.7×10 11 cps/mg protein) was recovered, and the activity recovery rate was 81% (total recovery rate: 45%).

100 ml of the active fraction was desalted using Sephadex G-25 (carrier: 5,000 ml, manufactured by Pharmacia) using 20 mM Tris-HCl buffer (pH 7.1) as a developing solution. This was mixed with 20mM Tris-HCl buffer (pH
It was adsorbed onto a heparin cellulofine column (carrier: 50 ml, manufactured by Seikagaku Corporation) equilibrated with 7.1).

The column was then washed with the same buffer used for equilibration, and the luciferase was eluted with a linear NaCl gradient from 0M to 1M. 8.5x1011cp
s (specific activity: 1x1013cps/mg prote
in) luciferase was recovered, and the activity recovery rate was 65
% (total recovery rate: 29%). This purified fraction showed a single band in SDS electrophoresis under reducing conditions, and was confirmed to be highly pure luciferase.

Example 2 The recombinant yeast of Example 1 was cultured in a 400 L culture tank, and the bacterial cells were removed by centrifugation to obtain 195 L of supernatant. Luciferase activity is 2.7x1013cps in total (specific activity: 1.3x109cps/mg protein)
Met. This supernatant was adsorbed on DEAE Cellulofine A500 (carrier: 5.5 L, manufactured by Seikagaku Corporation) equilibrated in advance with 20 mM Tris-HCl buffer (pH 7.1). The column was washed with equilibration buffer and the luciferase was then eluted with a linear gradient of 0M to 0.3M NaCl. 1.7x1013cps (specific activity: 1.
6 x 1010 cps/mg protein) of luciferase was recovered, and the activity recovery rate was approximately 59%.

Butyl Toyopearl 650 was prepared by adding 30% saturated ammonium sulfate to 3.5 L of the active fraction and equilibrating it with 20 mM Tris-HCl buffer (pH 7.1) containing 1 M ammonium sulfate.
It was adsorbed onto M (carrier: 1.0 L). The column was washed with the buffer used for equilibration (containing 1M ammonium sulfate), and luciferase was eluted with a linear concentration gradient of ammonium sulfate from 1M to 0M. 1.5x1013cps (specific activity: 1.7x1
011 cps/mg protein) of luciferase was recovered. The activity recovery rate was about 88% (total yield: about 56%).

[0021] 550 ml of the obtained active fraction was diluted with 10 mM
After desalting and buffer replacement with Sephadex G-25 (carrier: 5.0 L, manufactured by Pharmacia) using sodium phosphate buffer (pH 7.1) as a developing solution, the solution was adjusted to 0.5M. NaCl was added. Add this to 0.5M NaC
10mM sodium phosphate buffer (pH 7.1) containing
) equilibrated with a copper chelate sepharose column (support:
After adsorption in 120 ml (manufactured by Pharmacia), it was washed with the same buffer (containing 0.5M NaCl) used for equilibration. Luciferase was then eluted with a linear gradient of NH4Cl from 0M to 0.3M. 1.3x1013c
ps (specific activity: 1.0x1013cps/mg pr
otein) luciferase was recovered. The activity recovery rate was about 87% (total yield: about 48%). This purified fraction showed a single band in SDS electrophoresis under reduced conditions, and was confirmed to be highly pure luciferase.

[0022]

[Effects of the Invention] According to the present invention, Cypridina luciferase, which has conventionally been difficult to produce industrially, can be produced with high purity and efficiency.

Claims (1)

[Claims] [Claim 1] A Cypridina luciferase gene, which is characterized in that a culture solution of a recombinant Cypridina luciferase gene is purified by one or more of ion-exchange chromatography, hydrophobic chromatography, and affinity chromatography. Method for producing luciferase.