JPH11239480A - Recombinant dna connecting dna encoding prenyl 2-phosphoric acid synthase and production of prenyl 2-phosphoric acid synthase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new recombinant DNA useful for effective production, etc., of prenyl-2-phosphoric acid synthase by connecting DNA encoding prenyl-2-phosphoric acid synthase to the downstream of a promoter region derived from Bacillus brevis. SOLUTION: This new recombinant DNA links DNA encoding prenyl-2- phosphoric acid synthase to the downstream of a promoter region derived from Bacillus brevis and is useful for efficient production, etc., of prenyl-2-phosphoric acid synthase used for applying to research, diagnosis, treatment and industrial materials. The recombinant DNA is obtained by adding a signal sequence containing NcoI to 5' end of a gene containing DNA sequence encoding prenyl-2- phosphoric acid synthase and adding a part of a synthetic DNA containing Hind III site to the 3' end of the above gene to prepare a DNA for vector introduction and inserting the DNA into plasmid vector pNV212 containing erythromycin-resistant gene derived from Bacillus brevis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a prenyl diphosphate synthase.

[0002]

2. Description of the Related Art With the development of gene recombination technology, many proteins have been expressed as recombinant proteins by various expression systems including bacteria, fungi and mammals. However, in the case of using a host of intracellular expression, which is a bacterium generally used for expressing a heterologous protein, a problem often arises.

[0003] For example, when an Escherichia coli system is used, the produced heterologous protein accumulates in the cells and often forms cell inclusions. The heterologous protein forming the inclusion body is biologically or biochemically inactive, and solubilization and regeneration operations are required to obtain an active protein. And if the operation of solubilization and regeneration is not successful,
The required amount of active protein cannot be expected. However, a universal method of solubilization and regeneration has not been established yet, and much trial and error is required.

[0004] Even when a heterologous protein does not form an inclusion body, the heterologous protein produced and accumulated in the cells requires a great deal of time and effort for extraction from the cells and purification from the extract. In addition, in some cases, the target protein cannot be obtained purely in perfect form. For example, among prenyl diphosphate synthases, there has been no report that geranylgeranyl diphosphate synthase could be purified solely without devising a fusion protein with another protein.

[0005] Among bacterial systems, microorganisms belonging to the genus Bacillus have been industrially used since ancient times as bacteria producing various extracellular enzymes. Among these extracellular enzymes, the α-amylase gene of Bacillus amyloliquefaciens [I. Palva et. al, Gene, 22, 22
9 (1983)], the Bacillus licheniformis penicillinase gene and the Bacillus subtilis α-amylase gene have already been cloned, and an extracellular secretory production system for heterologous proteins using their promoters and enzyme signal peptides has been reported. ing. In this extracellular secretory production system, the formation of inclusion bodies, which can be a problem in the above-described intracellular expression system, is hardly observed. Therefore, production costs can be significantly reduced.

[0006] There are no reports on the production technology of recombinant prenyl diphosphate synthase in an extracellular expression system currently used in the art, and it is intended for use in research, diagnosis, treatment and industrial material applications. There remains a need for superior production methods for the production and purification of prenyl diphosphate synthase.

[0007]

In the expression system of a foreign protein such as prenyl diphosphate synthase, there are problems such as complexity of enzyme solution preparation and expression amount, formation of inclusion bodies, and stability in cells. However, none of the currently reported production systems are satisfactory. Accordingly, an object of the present invention is to provide a novel method capable of efficiently producing a prenyl diphosphate synthase by secreting the enzyme into a medium.

[0008]

Means for Solving the Problems The present inventors have developed a recombinant secretion expression system capable of stably expressing a heterologous protein in a medium in a recombinant expression system. On the other hand, studies on establishment of a recombinant expression system capable of efficiently secreting and expressing prenyl diphosphate synthase, which cannot be expected to extracellularly secrete without special measures because it is an enzyme originally present in cells, were repeated. -It has been found that efficient expression is possible in the secretory production system of Brevis.

Therefore, according to the present invention, in a certain host cell in which a limited amount of protein is normally secreted into the medium, a large amount of prenyl diphosphate synthase having a desirable tertiary structure is secreted into the medium. The present invention provides a DNA encoding a peptide, a host cell retaining the DNA, and a method for producing a prenyl diphosphate synthase, which comprises culturing the host cell.

[0010]

According to the present invention, a stable and soluble prenyl diphosphate synthase can be produced in a large amount in a certain kind of host cell in a medium. Construction of DNA encoding prenyl diphosphate synthase
DNA encoding prenyl diphosphate synthase selected by method
For example, the sequence of farnesyl diphosphate synthase is disclosed in Japanese Patent Application No. 3-253788 (JP-A-5-219).
961) and geranylgeranyl 2
As the phosphate synthase, the sequences described in Japanese Patent Application No. 6-315579 (Japanese Patent Application Laid-Open No. 7-308193) can be used. Construction of the plasmid for expression of the prenyl diphosphate synthase of the present invention employs general genetic engineering techniques in the art [Sambrook et al., "Molecular Cloning, A Laboratory Manual", Cold Spring・ Harbor Laboratory,
Cold Spring Harbor, New York (19
88)].

Expression vector DN encoding prenyl diphosphate synthase as described above
The A molecule can carry other sequences for heterologous protein expression according to the invention. Desirable DN according to the invention
Examples of the A sequence include various DNA sequences under the control of a promoter sequence for transcription in a host cell. For example, when the host cell is Bacillus brevis, the DNA molecule contains a promoter and a ribosome binding site that function in Bacillus brevis, and also contains a secretory signal sequence for extracellular movement of the protein.

[0012] If necessary, the gene may contain a selectable marker gene. If the DNA molecule exists outside the chromosome in the host cell, it may contain a replication origin.
Specific sequences that function in Bacillus brevis are described in Methods in Enzymology, vo.
l. 217, p23-33 (1993)). Expression vectors used for bacterial host expression are also known, as well as expression vectors for many bacterial hosts, including the components described above, are known in the art and can be used in standard molecular biology applications. It can be easily constructed by technology.

D containing the prenyl diphosphate synthase gene
The NA molecule can be modified to the most appropriate codon for translation among the codons encoding the same amino acid to maximize expression in the selected host cell, as is commonly done in the art. Multiple copies of the prenyl diphosphate synthase gene may be present in one DNA molecule.

Host The host cell suitable for the present invention is preferably a bacterium. Bacillus brevis, well known in the art as a host cell for secretory expression, is known by Udaka et al.
methods in Enzymology, vol.
217, P23-33 (1993)), and the Bacillus brevis strain 31-OK used in the following examples have been deposited with the National Institute of Advanced Industrial Science and Technology as FERMP-13274. Also, various strains such as Bacillus and Pseudomonas including Bacillus subtilis can be used in the present invention.

[0015]

The following is an example relating to the production of prenyl diphosphate synthase. (1) Design of DNA encoding prenyl diphosphate synthase The DNA sequence of prenyl diphosphate synthase is described in Japanese Patent Application No. 3-253788 for farnesyl diphosphate synthase.
(Japanese Unexamined Patent Publication (Kokai) No. 5-219661) and geranylgeranyl diphosphate synthase are disclosed in Japanese Patent Application No. Hei.
315579 (JP-A-7-308193).

A part of the signal sequence containing NcoI at the 5 'end of the gene and a HindIII site at the 3' end were added with synthetic DNA to synthesize DNA for vector introduction. The synthesized gene was transformed into the vector pT7Blue-T.
(Purchased from Novagen) and introduced into E. coli HB1
01 was transformed, a plasmid was extracted from the obtained clone, and the nucleotide sequence was determined by the dideoxy method to confirm that there was no mutation.

(2) Introduction of prenyl diphosphate synthase gene into Bacillus brevis expression vector A prenyl diphosphate synthase gene fragment was cut out from the constructed plasmid with NcoI and HindIII. This was transformed into a plasmid vector pNU212 (Methods in Enzy) containing the erythromycin resistance gene.
molology, vol. 217, pp. 23-33 (199
3) DNA inserted into NcoI and HindIII sites
It was ligated with ligase to obtain an expression plasmid vector.
(Fig. 1)

(3) Obtaining Transformants Transformation was performed according to the method described by Takagi et al. In Agric. Biol. Che
m. 53, 3099-3100, (1983). PNU constructed
About 500 ng of the 212-based expression vector was used to transform Bacillus brevis strain 31-OK.

(4) Expression and analysis of prenyl diphosphate synthase The obtained transformant was transformed into YC medium [30 g polypeptone P
1 (Nippon Pharmaceutical), 2g Yeast Extract, 30g
Glucose, 0.1 g calcium chloride dihydrate, 0.1
g magnesium sulfate heptahydrate, 10 mg iron sulfate heptahydrate,
The mixture was cultured at 30 ° C. for 3 days with manganese sulfate heptahydrate, pH 7.2 per 1 mg zinc sulfate tetrahydrate / l.

The protein contained therein was separated by SDS-PAGE (12%) using 10 μl of the culture supernatant, and visualized by CBB staining, or immobilized on a nitrocellulose membrane by Western blotting. As a primary antibody used for immunostaining for visualizing prenyl diphosphate synthase after Western blotting, antiserum obtained by immunizing rabbits with various prenyl diphosphate synthases was used.

After allowing the blotted nitrocellulose filter to react with the primary antibody, an alkaline phosphatase-labeled anti-rabbit IgG antibody was used as a secondary antibody to form a B antibody.
Detection was performed using CIP / NBT (Bio-Rad color development kit). FIG. 2 shows a diagram when the expression of each prenyl diphosphate synthase was confirmed by Western blot. It was confirmed as a single band at the position of the target molecular weight. The expression level when pNU212 was used as the expression vector was about several tens of mg / L.

(5) Confirmation of Activity of Expressed Prenyl Diphosphate Synthase 5-1. Enzyme sample The culture supernatant of each clone was used as an enzyme sample. 5-2. Reaction solution The composition of the reaction solution was as follows. 0.5 M bis Tris (9.5) 20.0 μl 0.3361 mM IPP (1 Ci / mol) 74.4 μl 0.5 mM DMAPP 50.0 μl 0.1 M MgCl ₂ 10.0 μl Sample 10.0 μl H ₂ O 35.6 μl

5-3. Activity measurement Activity measurement was performed as follows. After reacting at 70 ° C. for 30 minutes, 50 μl of 0.5 M EDTA and 150 μl
NaCl was added, butanol saturated with water was further added, and the mixture was stirred, followed by centrifugation, and the radioactivity in the butanol layer was measured. As a result, the prenyl diphosphate synthase produced by Bacillus brevis had an activity equivalent to that of the original enzyme, and the production amount was the same as that calculated by the electrophoresis method.
It was 0 mg / L.

[Brief description of the drawings]

FIG. 1 is a diagram showing the construction of a prenyl diphosphate synthase diagram expression vector of the present invention.

FIG. 2 shows geranylgeranyl diphosphate synthase (GGPS) and farnesyl diphosphate synthase (FP).
S) A photographic diagram showing electrophoresis showing the result of detecting an expression product from a culture solution of Bacillus brevis having an expression plasmid by Western blotting.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/21 C12R 1:08) (C12N 9/12 C12R 1:08) (72) Inventor Haruo Takahashi Nagakute, Aichi-gun, Aichi Prefecture 41, Chuo-cho, Chumachi, Yokomichi, Toyoda Central Research Laboratory Co., Ltd. (72) Inventor Masanori Hirai 41, Chuo-chuk, Yoji, Nagakute, Aichi-gun, Aichi, Japan Toyota Chuo Research Institute, Inc. (72) Inventor Yamada Yukio 41-41, Chuchu-Yokomichi, Nagakute-cho, Aichi-gun, Aichi Prefecture Inside Toyota Central Research Laboratory, Inc. (72) Inventor Masayoshi Muramatsu 1 Toyota Town, Toyota City, Aichi Prefecture Inside Toyota Motor Corporation (72) Inventor Ohne Toku 1 Toyota Town, Toyota City, Aichi Prefecture Inside Toyota Motor Co., Ltd. (72) Inventor Un ▲ Taka ▼ Shigezo 1-34 Ueno-cho, Meito-ku, Nagoya City, Aichi Prefecture

Claims (3)

[Claims] 1. Bacillus brevis (Bacillus)
brevis), to which a DNA encoding prenyl diphosphate synthase is linked downstream of a promoter region. 2. A host cell capable of secreting and expressing a foreign protein by introducing the recombinant DNA according to claim 1 into the cell. 3. Production of prenyl diphosphate synthase, wherein the host cell according to claim 2 is cultured in a nutrient medium, and the prenyl diphosphate synthase produced thereby is collected from the culture. Method.