JP6386029B2 - Cypridina luciferase production method - Google Patents

Description

[Cross-reference of related applications]
This application claims priority based on Japanese Patent Application No. 2014-076936 filed on Apr. 3, 2014, the entire disclosure of which is incorporated herein by reference.

  The present invention relates to a method for producing and purifying Cypridina luciferase and Cypridina luciferase fusion protein.

  The luminescence quantum yield of bioluminescence such as luciferin / luciferase reaction is significantly higher than that of chemiluminescence, and is considered suitable for trace analysis such as enzyme immunoassay. In particular, the Cypridina luminescence system has attracted attention as a highly sensitive detection system for enzyme immunoassay. Cypridina luminescence system has a high quantum yield and the number of rotations of the enzyme is more than 10 times that of firefly luciferase and the like, and applied research to enzyme immunoassay has been conducted (Patent Documents 1 to 4). ). An inexpensive and effective Cypridina luciferase modification method suitable for enzyme immunoassay has also been developed (Patent Document 5). However, in the method for producing Cypridina luciferase, in the system using the yeast of Non-Patent Document 1, yeast secretes a large amount of Cypridina luciferase, but also secretes many proteins at the same time, and there are also proteins contained in the medium. However, the purification is not easy, and it is purified through a three-step purification process. Non-patent document 2 discloses a production method using insect cell silkworms, but even affinity chromatography has a purity of about 95% and is not suitable for highly sensitive measurement. In addition, when Cypridina luciferase is produced using Escherichia coli instead of these insect cells and yeast, there is almost no luciferase activity. Thus, the Cypridina luminescence system has attracted attention as a highly sensitive detection system for enzyme immunoassay, and processes such as biotinylation have become highly efficient. Not established.

JP 5-64583 JP 5-113443 JP 7-98316 A JP-A-8-262021 Patent No. 4911408

Wu C, et al., Analytical Chemistry 79: 1634-1638, 2007 Wu C, et al., Proc Natl Acad Sci U S A. 106 (37): 15599-603, 2009 Plant Biotechnology, 2011 28, 201-210 Plant Biotechnology Journal, 2006 4, 325-332

  The problem to be solved by the present invention is to provide a technique for producing Cypridina luciferase with high efficiency and high purity.

  Under the circumstances as described above, the present inventors have intensively studied, and as a result, among various cells, by using plant cells rather than insect cells, yeasts and the like that have been conventionally used for the production of Cypridina luciferase, It has been found that cypress firefly luciferase can be produced efficiently and with high purity. The present invention is based on such novel findings.

  Since plant cells take time to establish cells and the cells cannot be cryopreserved, they have not been used so far in substance production. In addition, since plant cells have cell walls, there is a problem that it is difficult to secrete the target substance out of the cell even if the production is attempted by introducing the gene of the target substance into the plant cell. However, as a result of studying a wide variety of cells as host cells, the present inventors have used plant cells for the production of Cypridina luciferase. Surprisingly, Cypridina luciferase produced in the cell is secreted outside the cell. Therefore, it was found that the obtained protein can be purified very easily.

  Furthermore, as described above, the activity of Cypridina luciferase obtained by the type of host cell is different, and it is very difficult to predict the level of the activity. Under such circumstances, the present inventors have examined various cells, and as a result, the sea urchin luciferase obtained using plant cells is not only easy to purify but also surprisingly very active and closer to natural products. I found out that it was.

Accordingly, the present invention aims to provide the following inventions:
Item 1. A method for producing Cypridina luciferase, which comprises a step of introducing a Cypridina luciferase gene into the genome of a plant cell.

  Item 2. Item 2. The method according to Item 1, wherein the gene introduction step is performed using Agrobacterium.

Item 3. Item 3. The method according to Item 1 or 2, further comprising the step of culturing the plant cell obtained in the gene introduction step and the step of recovering Cypridina luciferase secreted by the plant cell.

  Item 4. Item 4. The method according to Item 3, further comprising the step of selecting a strain having a Cypridina luciferase activity among the plant cells obtained in the culturing step.

Item 5. Item 5. The method according to Item 3 or 4, which comprises a step of purifying Cypridina luciferase obtained in the recovery step.

  Item 6. Item 6. The method according to any one of Items 1 to 5, wherein the plant is tobacco.

  Item 7. Plant cells into which the sea urchin luciferase gene has been introduced.

  Item 8. Item 8. The plant cell according to Item 7, wherein the plant is tobacco.

  According to the present invention, highly pure Cypridina luciferase that can be used in immunoassays can be produced efficiently and simply. In addition, a complex of Cypridina luciferase obtained by the method of the present invention and avidin, streptavidin, neutravidin, etc. can also be prepared, and immunoassay (especially enzyme immunity such as direct method, indirect method, sandwich ELISA method, ELISPOT method, etc.) Measurement method), DNA probe method, or various assays such as receptors, ligands and sugar chains, particularly assays for quantifying proteins and DNA.

This shows the amino acid sequence of AAB86460 (SEQ ID NO: 1). The amino acid sequence of AAA30332 (SEQ ID NO: 2) is shown. The amino acid sequence of BAD08210 (SEQ ID NO: 3) is shown. This shows the base sequence of the Cypridina luciferase gene (SEQ ID NO: 4). The base sequence (SEQ ID NO: 7) of the firefly luciferase gene is shown. The base sequence (SEQ ID NO: 8) of the Cypridina luciferase gene for yeast expression is shown. The nucleotide sequence of the Renilla luciferase gene (SEQ ID NO: 9) is shown. The outline | summary of a reference example of this application and the result of the activity measurement of the firefly luciferase expressed in the leaf of Arabidopsis thaliana and the sea urchin luciferase are shown. The result of the activity measurement of Cypridina luciferase produced using tobacco cultured cells and silkworm cells is shown. The distribution of Cypridina luciferase activity in the cell mass obtained in the culturing step in Examples of the present application is shown. The result of measuring the activity of untagged or tagged Cypridina luciferase produced using tobacco cultured cells is shown. The graph which shows the relationship between the substrate density | concentration of natural cypress firefly origin luciferase measured in Example 3, and emitted light intensity and an approximated curve are shown. The graph and the approximate curve which show the relationship between the substrate concentration of the cultured cell production Cypridina luciferase measured in Example 3, and luminescence intensity are shown. The graph and the approximate curve which show the relationship between the substrate concentration of the cultured cell production Avi tag fusion luciferase measured in Example 3, and luminescence intensity are shown.

  In the present invention, “protein (protein)” and “peptide” are used to include oligopeptides and polypeptides. In the present specification, “protein” and “peptide” include both a protein modified with a sugar chain and an unmodified protein unless otherwise specified. The same applies to proteins that are not specified as proteins.

  In the present invention, “gene” includes double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof unless otherwise specified. In the present invention, “gene” refers to a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified.

Method for Producing Cypridina Luciferase The present invention provides a method for producing Cypridina luciferase using plant cells.

  Cypridina luciferase used in the present invention is publicly known, and examples thereof include luciferase derived from Cypridina noctiluca (Cypridina luciferase), luciferase derived from Vargula genus Cypridina (Vargula luciferase), and Cypridina luciferase is preferable. . In the present specification and claims, unless otherwise indicated, the term “cypridina luciferase” broadly encompasses wild-type Cypridina luciferase, as well as any variant thereof having luciferase activity and fusions thereof. Examples of the amino acid sequence of wild-type Cypridina luciferase include those described in the NCBI database under access numbers AAB86460 (SEQ ID NO: 1), AAA30332 (SEQ ID NO: 2), BAD08210 (SEQ ID NO: 3) and the like. The amino acid sequences of SEQ ID NOs: 1 to 3 are shown in FIGS.

  Cypridina luciferase is one or more, preferably one or several amino acids may be substituted, added, deleted or inserted, and any variant as long as it has the activity of emitting cypridina luciferin as a substrate Is included.

  Examples of Cypridina luciferase fusions include those having peptides, proteins, etc. having affinity for other proteins at the C-terminus, or proteins having other functions such as fluorescence, etc. Any fusion is included as long as it has the activity of emitting light as a substrate. Cypridina luciferase fusions include those obtained by adding His tag, avidin tag, streptavidin tag, neutravidin tag, etc. to Cypridina luciferase.

  Also, for example, a modified Cypridina luciferase is biotinylated at a predetermined position of Cypridina luciferase (lysine residue at a predetermined position, etc.), optionally via a polyalkylene glycol structure (polyethylene glycol, etc.) as a spacer. Things are also included. The modified Cypridina luciferase includes, for example, those in which a signal peptide is substituted or added so as to be suitable for secretion from plant cells.

  In the present invention, plant cells typically mean cells collected from plants (plant-derived cultured cells and the like).

  The method according to the present invention may include a step of expressing Cypridina luciferase using plant cells. For this step, for example, a method of transforming a plant cell with a Cypridina luciferase gene and expressing the Cypridina luciferase gene with a transformant can be widely used. In the present invention, examples of the Cypridina luciferase gene include nucleic acid molecules encoding the Cypridina luciferase shown above.

  The plant cell used in the present invention may be a plant-derived primary cultured cell or a cell line. As plant cells, cells of higher plants are preferred. Here, examples of higher plants include angiosperms and gymnosperms, and angiosperms are preferred. Examples of angiosperms include dicotyledonous plants and monocotyledonous plants. More specific examples of plant cells are not particularly limited, and examples include tobacco, rice, Arabidopsis, grapes, tomatoes, etc., tobacco (BY2 cells, BY2-H cells, 3n-3 cells, NT1 cells, etc.) Etc.) are preferred.

  The method of the present invention includes the step of introducing a Cypridina luciferase gene into the genome of a plant cell. In the present invention, “introducing a gene into a genome” can be rephrased as “incorporating into a chromosome”. As such a gene introduction method, a known method suitable for the use after introduction can be selected and carried out. Specific examples of the method include an Agrobacterium method, a particle gun method, an electroporation method, and a PEG method. The Agrobacterium method and the like are preferable from the viewpoint of easy introduction of the Cypridina luciferase gene into the genome. When the gene introduction step is performed using Agrobacterium, for example, a Cypridina luciferase gene can be introduced into Agrobacterium, and Agrobacterium into which the gene has been introduced is infected with plant cells. When the Agrobacterium method is used, examples of Agrobacterium include Agrobacterium tumefaciens (GV3101, C58C1, GV2260, LBA4404, EHA101, EHA105, etc.) and the like.

  In a preferred embodiment of the present invention, the gene introduction step can be performed using a vector. For example, the above-described step of introducing the Cypridina luciferase gene into Agrobacterium can be performed using a vector. More specifically, a method of introducing a vector having a Cypridina luciferase gene into Agrobacterium and infecting plant cells with Agrobacterium into which the vector has been introduced can be mentioned. The plant cell into which the Cypridina luciferase gene has been introduced by the method of the present invention has the Cypridina luciferase gene integrated into the genome, and thus stably retains the gene.

  Examples of the vector include p35SG / pBCKH, pDEST_35S_HSP_GWB5, and pBI101. The Cypridina luciferase gene may be placed under the control of a promoter in the vector. The promoter is not particularly limited, and examples thereof include CaMV35S, UBQ1, and ACT1. The vector containing the mutant luciferase gene of the present invention includes a multiple cloning site, a promoter sequence, an enhancer sequence, a polyadenylation sequence, an insulator sequence, a selectable marker gene sequence, a replication for introducing the mutant luciferase gene as necessary. It may have a starting point. Enhancer sequences include tobacco mosaic virus (TMV) omega translation enhancer, tobacco etch virus (TEV) translation enhancer, and 5'UTR region of Arabidopsis ADH1.

  The present invention may include a step of culturing a plant cell into which a vector having a Cypridina luciferase gene has been introduced. As the medium used in the method of the present invention, those used for culturing can be widely used depending on the kind of plant cells, and examples thereof include MS medium and B5 medium. The culture temperature is not particularly limited, and can be appropriately set, for example, in the range of 20 to 30 ° C, preferably 22 to 28 ° C. The culture time is not particularly limited, and can be appropriately set in the range of, for example, 24 to 480 hours, preferably 240 to 360 hours. By carrying out the above culture, Cypridina luciferase is secreted from the plant cells.

  In the method of the present invention, a step of recovering Cypridina luciferase obtained in the expression step may be performed. There are no particular limitations on the recovery method, and techniques commonly used in the field of protein production can be widely employed.

  In the method of this invention, you may perform the process of selecting the strain | stump | stock which has Cypridina luciferase activity among the plant cells obtained at the said culture | cultivation process. At that time, the Cypridina luciferase activity can be measured, for example, by the method described in the Examples of the present application. In the selection step, it is preferable to select, for example, a strain having high Cypridina luciferase activity from the plant cells obtained in the culturing step. In the step of selecting a strain having high Cypridina luciferase activity, typically one strain or a plurality of strains (a group of strains) having the highest Cypridina luciferase activity are selected. In the present invention, the “step for selecting a strain having high Cypridina luciferase activity” can also be called “the step for selecting a strain having a predetermined Cypridina luciferase activity”. Further, in the method of the present invention, a step of further selecting one or more strains obtained with a homogeneous culture solution and / or having a growth rate of a certain level or more with respect to the strains selected in the above selection step. This may be done optionally.

  In the method of the present invention, the sea urchin luciferase can be obtained with high purity by using plant cells. However, the recovered sea urchin luciferase may optionally be further purified.

  The protein purification method is not particularly limited, and examples thereof include removal of contaminants by a filter, gel filtration, chromatography (ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, normal phase chromatography). Chromatography), electrophoresis, precipitation methods (ammonium sulfate precipitation, precipitation with organic solvents, isoelectric point precipitation, etc.) and the like.

  The Cypridina luciferase obtained by the method of the present invention can be used in the state of a cell culture solution, in the state of a cell lysate, used as a culture supernatant, or purified from the culture supernatant by the above method or the like. You may use what you did.

Plant Cell The present invention provides a plant cell into which a Cypridina luciferase gene has been introduced. Such plant cell types and production methods are as described above. The plant cell of the present invention can secrete highly pure Cypridina luciferase into the medium.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to this Example.

Reference Example First, in order to confirm whether Cypridina luciferase functions normally in plants, an experiment was conducted to measure luminescence by transiently expressing Cypridina luciferase gene in Arabidopsis thaliana using a particle gun. . The p35SHSPG vector described in Non-Patent Document 3 was cleaved with SmaI, and the Cypridina luciferase gene (NCBI access number AB177531) represented by SEQ ID NO: 4 was amplified by PCR using the following oligonucleotide pair as a primer and inserted (35S : CLUC). The base sequence of the Cypridina luciferase gene shown in SEQ ID NO: 4 is shown in FIG.
5'-GATGAAGACCTTAATTCTTGCCGTTG-3 '(SEQ ID NO: 5)
5'-CTATTTGCATTCATCTGGTACTTCT-3 '(SEQ ID NO: 6)
Similarly, a plasmid for expressing the firefly luciferase gene (SEQ ID NO: 7) was also prepared as a comparative control (35S: FLUC). In addition, a plasmid for expressing the luciferase gene (SEQ ID NO: 8) derived from the sea urchin for yeast expression described in the NCBI database under the access number AB259056 was also prepared (35S: CLUCY). In the experiment, the Renilla luciferase gene (SEQ ID NO: 9) expressed by the CaMV35S promoter was used as an internal standard (35S: RLUC). Gold particles were coated with 35S: CLUC (or 35S: FLUC) and 35S: RLUC plasmids, and then shot into rosette leaves of Arabidopsis thaliana with a stationary particle gun. Thereafter, the mixture was allowed to stand for 16 hours in a dark place at 23 ° C., and the tissue was crushed in a buffer solution to extract proteins. A substrate solution of each luciferase was added to a part of the protein extract, and the luminescence intensity was measured with a luminometer.

The conditions for activity measurement are as follows:
Mix 1 microgram / microliter of each enzyme solution 1000-fold diluted with phosphate buffered saline with 20 microliters of 2 micromolar Cypridina luciferin solution and immediately mix them with phelios (manufactured by ATTO). Counting was performed for 10 seconds, and the integrated value was used as a relative activity value.

  As a result, as shown on the right side of FIG. 8, it was found that the sea urchin luciferase emits light in the plant without much difference from the firefly luciferase.

Example 1
To investigate whether Cypridina luciferase is also secreted extracellularly in plants, and in that case, whether the plant is suitable for Cypridina luciferase production, the Cypridina luciferase gene was transferred to tobacco BY2 cultured cells (Toshiyuki Nagata, Yasuyuki Nemoto , and Seiichiro Hasezawa, International Review of cytology, vol.132, pp 1-30 (1992)). First, 35S: CLUC prepared in Reference Example was transferred to the pBCKK vector described in Non-Patent Document 4 by gateway LR reaction. This plasmid was introduced into the soil bacterium Agrobacterium GV3101 (Agrobacterium tumefaciens strain GV3101 (C58C1Rifr) pMP90 (Gmr) (koncz and Schell 1986)) strain by electroporation. The introduced bacteria were cultured in 2 ml of LB medium containing kanamycin for 1 day. On the other hand, 4 ml of BY2 cultured cells 3 days after transplanting were collected, and acetosyringone was added to a final concentration of 20 micromolar. Thereafter, pipetting was performed 20 times with a 10 ml measuring pipette to give fine scratches to the cells. Next, 100 microliters of Agrobacterium solution cultured for 1 day was added and co-cultured for 3 days at 25 ° C. in the dark. Three days later, the plate was washed repeatedly with a liquid medium for BY2 containing kuraforan, spread on an agar medium for BY2 containing kanamycin and kraforan, and cultured at 25 ° C. The cell mass that grew after 2 or 3 weeks was repeatedly planted on a new agar medium, and the luciferase activity was measured in the same manner as in the Reference Example for the cell mass grown to a certain size. Among them, several lines with particularly high activity were transplanted into a BY2 liquid medium to form cultured cells. After repeating the subculture once a week, the culture supernatant was collected from the cultured cells for 2 weeks after transplanting, and the luciferase activity was measured, and the Cypridina luciferase protein was confirmed by SDS-PAGE and CBB staining. .

  In addition, as a comparative example, CLUC luciferase was produced using a silkworm cell and a silkworm nuclear polyhedrosis virus in accordance with the method described in Japanese Patent No. 3090586, and the activity was measured.

  The results are shown in FIG. As a result, it was found that Cypridina luciferase is secreted into the medium from tobacco BY2 cultured cells and stably present in the medium. As a result of electrophoresis, two bands were observed near 70 kDa, but no other prominent protein could be confirmed (FIG. 8, right). Until now, Cypridina luciferase synthesized in silkworms has also been confirmed as two bands, so the proteins in these two bands are Cypridina luciferases with slight differences in folding and must be purified for immunoassay. A certain level of uniformity is guaranteed. Centricon (protein concentration filter) was used to remove low molecular weight compounds, and concentration and buffer exchange were easily achieved. A purified product of about 20-30 mg per 1 L medium was obtained. This is the same level of expression as the yeast cell system used in Non-Patent Document 3, but the yeast cells undergo a three-stage purification to achieve the same degree of purification, whereas tobacco cells have a complex column. It becomes possible to omit purification.

  As described above, a high-purity protein with a purity of 98% or more could be produced at a level of 20-30 mg per liter by removing low molecular weight compounds with a filter. Therefore, highly efficient and easy-to-purify Cypridina luciferase can be produced easily and efficiently by using plant cells, and an enzyme immunization method using Cypridina luciferase can be easily constructed.

  The activity of purified Cypridina luciferase is shown on the left side of FIG. As shown in the left graph of FIG. 9, the activity of the sea urchin luciferase produced using tobacco cells is the same as that of the sea urchin luciferase produced using silkworm cells, although the content of the sea urchin luciferase in the enzyme solution is the same. Was significantly higher than.

Example 2
The p35SHSPG vector described in Non-Patent Document 3 was cut with SalI and SacI, and DNA annealed with the following oligonucleotide pairs was inserted to create a p35S_avi_HSPG vector.
5'-TCGACTCCGGCTTGAACGACATCTTCGAGGCCCAGAAGATCGAGTGGCACGAGTAGAGCT-3 '(SEQ ID NO: 10)
5'-CTACTCGTGCCACTCGATCTTCTGGGCCTCGAAGATGTCGTTCAAGCCGGAG-3 '(SEQ ID NO: 11)
Next, this vector was cut with SmaI, and the Cypridina luciferase gene was amplified and inserted by PCR using the following oligonucleotide pair as a primer (35S: CLUC-avi).
5'-GATGAAGACCTTAATTCTTGCCGTTG-3 '(SEQ ID NO: 5)
5'-TTTGCATTCATCTGGTACTTCT-3 '(SEQ ID NO: 12)
Next, a gateway LR reaction is performed with the pBCKK vector for plant transformation described in Methods in Molecular Biology, 2011 754, 87-105, and parts other than the vector backbone are transferred to the pBCKK vector, and soil bacteria A secretory expression system was established by transforming BY2 cultured cells using Agrobacterium. Here, FIG. 10 shows the activity distribution when luciferase activity was measured for each of the cell masses 166 clones grown to a certain size according to the reference example. Liquid culture was performed on 8 clones showing the highest activity values. Then, clones were obtained which obtained a homogeneous culture solution and confirmed a growth rate above a certain level. The cultured cell supernatant containing the Avi-tagged Cypridina luciferase obtained above is collected, and using HiTrapQ HP (manufactured by GE Healthcare Japan), 20 mM Tris-HCl (pH 8.0) buffer and 20 mM Tris-HCl (pH 8.0) The culture supernatant was fractionated by a gradient with 1 M NaCl buffer. Similarly to the reference example, the activity of each fraction was measured, and the fraction with high activity was collected. For comparison, Cypridina luciferase with no tag added was also recovered. Specifically, Cypridina luciferase without a tag was obtained by performing the same operation as described above except that 35S: CLUC was used instead of 35S: CLUC-avi. Each 100 ng equivalent was confirmed by SDS-PAGE (left of FIG. 11). Moreover, it used for activity measurement like the reference example using the solution which diluted 100 ng protein 10,000 times, respectively. It was confirmed that the activity was almost the same regardless of the presence or absence of the tag (FIG. 11 right).

Example 3
Affinity for Cypridina Luciferase Substrate Produced in Plant Cultured Cells Using Cypridina Luciferase Produced in Plant Cultured Cells, Reaction with a Stepwise Diluted Substrate (Citrus Luciferin) and Measuring the Luminescence Intensity, the Km Value Estimated experimentally. The Km value was estimated from the approximate curve for the Michaelis-Menten equation. Approximate curves as shown in FIGS. 12 to 14 for natural cypress firefly-derived luciferase, cultured cell-produced Cypridina luciferase obtained in Example 2 and cultured cell-produced Avi tag-fused luciferase obtained in Example 2 respectively. The obtained Km values were estimated to be 0.283 micromolar, 0.448 micromolar, and 0.395 micromolar from the approximate expression. From this result, it is considered that the plant cell-produced luciferase has the same substrate affinity as that of the natural type and is not affected at all by the addition of the tag.

Claims (5)

A method for producing Cypridina luciferase, comprising a step of introducing Cypridina luciferase gene into the genome of a plant cell ,
The gene introduction step is performed using Agrobacterium,
The plant is tobacco;
Way . The method according to claim 1, further comprising a step of culturing the plant cell obtained in the gene introduction step and a step of recovering the sea urchin luciferase secreted by the plant cell. The method of Claim 2 which further includes the process of selecting the strain | stump | stock which has Cypridina luciferase activity among the plant cells obtained by the said culture | cultivation process. The method of Claim 2 including the process of refine | purifying the Cypridina luciferase obtained at the said collection | recovery process. Tobacco cells into which the sea urchin luciferase gene has been introduced into the genome using Agrobacterium .

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