JPH084511B2 - Novel protein gene - Google Patents

Description

TECHNICAL FIELD The present invention relates to a gene, and more specifically, to a protein-producing bacterium Bacillus brevis.
vis) HPD31 protein, particularly a gene encoding a cell wall-derived extracellular protein.

(Prior Art) First, Utaka et al. Reported that the heat-resistant α- of Bacillus stearothermophilus DY-5.
PBAM10 with the amylase gene integrated in the plasmid pUB110
Bacillus brevis 47 and Bacillus subtilis possessing 1
Bacillus brevis 47 when cultured for about 15,000 U /
It was confirmed that approximately 3,000 U / ml of α-amylase was produced and accumulated in the culture medium for each of ml and Bacillus subtilis [J. Bacterio
L., 164, (3), 1182-1187 (1985)].

Utaka et al. Also reveal that the Bacillus brevis 47 strain contains two types of cell wall proteins (OWP, MWP), and also revealed the complete nucleotide sequences of these proteins [J.
Bacteriol., 168, (1), 365-373 (1986), J. Bacterio
L., 170, (2), 935-945 (1988)].

Certainly, the Bacillus brevis 47 strain created by Utaka et al. Is excellent in that it produces a significant amount of protein outside the cells, but it also has the property of producing proteolytic enzymes outside the cells. However, it has a drawback that the protein once produced is decomposed.

Therefore, Utaka et al. Succeeded in screening and isolating a new strain that produces a large amount of protein outside the cell but does not produce proteolytic enzyme outside the cell. This new strain was named Bacillus brevis H102 and deposited with the Institute of Microtechnology (FERM BP-1087) (JP-A-63-56277).

(Problems to be solved by the invention) This new bacterium Bacillus brevis H102 (FERM BP-1087)
Not only accumulates a very large amount of cell wall-derived protein of 30 g / l outside the cells, but also has a high ability to secrete heterologous gene products, and secretes 3 g / l of α-amylase derived from thermophiles. The remarkable usefulness was further discovered, and the bacteria were sorted out and H102 was renamed HPD31. Therefore, the H102 strain is hereinafter referred to as Bacillus brevis HPD31 strain.

The cell wall of B. brevis HPD31 consists of a protein layer and a peptidoglycan layer, and this cell wall constituent protein is 135K.
It consists of Da, which is called HWP or main protein.

Therefore, in view of the above-mentioned usefulness, there has been a strong demand in the art for development of a system for producing HPD31-derived protein in a larger amount.

Therefore, as a result of conducting various studies to meet the needs in the above industry, it has become necessary to elucidate the gene structure of a gene, particularly HWP.

(Means for Solving the Invention) The present invention has been made in order to achieve this object, and was made in order to clone the cell wall constituent protein gene of Bacillus brevis HPD31.

As a result of diligent research, we obtained the finding that HWP cross-reacts with the above antibody when western blotting was performed using an antibody of the B. brevis 47 MWP protein, indicating that the HWP gene and MWP gene have high homology. I expected.

And with this expectation, when the chromosomal DNA fragment of HPD31 was treated using the MWP gene already cloned by Utaka et al. As a probe, the HWP gene was successfully cloned, and the present invention was completed. is there.

 Hereinafter, the present invention will be described in detail.

That is, in the present invention, as a chromosomal DNA source, Bacillus brevis HPD31 (B. brevis H102) (FERM BP-
1087), a DNA fragment cleaved with a restriction enzyme or the like was incorporated into a phage, and a B. brevis 47-derived DNA fragment and probe A were used from among the phages incorporating these DNA fragments. Pick up the phage that has incorporated the HWP gene.

Next, a part of the DNA fragment thus obtained is used as probe B, and this is used to lift the HWP gene or main protein gene part of the HPD31-derived chromosomal DNA fragment.

Then, the obtained main protein DNA is ligated to a plasmid vector fragment using a DNA ligase or the like to prepare a recombinant DNA containing the gene of interest.

The chromosomal DNA derived from the HPD31 strain can be obtained by transforming this strain into, for example, T2 liquid medium (1% glucose, 1% peptone, 0.5% meat extract,
It can be obtained by shaking culture or aeration stirring culture with 0.2% yeast extract, pH 7.0) for about 1 to 3 days, collecting the obtained culture by centrifugation, and then lysing.

Known methods are appropriately used for this, for example, Saito, Mi
ura's method (Saito, H. and Miura, K., Biochim. Biophys.Ac
ta, 72 , 619, (1964)), the method of Rodoriguez et al. (Rodorigu
ez and Tait, Recombinant DNA Techniques (Addison−W
esleypub.Co) (1983)) is available.

Subsequent processing is performed by a known method, and the lysis method is
For example, treatment with a cell wall lytic enzyme such as lysozyme or β-glucanase, ultrasonic treatment, or the like is used. If necessary, other enzyme agents such as protease and ribonuclease and a surfactant such as sodium lauryl sulfate are used together. In addition, freeze-thaw treatment may be applied. In order to separate and purify DNA from the lysate thus obtained, by appropriately combining methods such as phenol extraction, deproteinization treatment, protease treatment, ribonuclease treatment, alcohol precipitation and centrifugation according to a conventional method. It can be carried out.

The method of cleaving DNA can be performed by, for example, ultrasonic treatment, restriction enzyme treatment, or the like. After cleavage, a modifying enzyme such as phosphatase or DNA polymerase is used if necessary. The nucleotide sequence at the end of the DNA fragment can be changed by using various linkers and adaptors.

From the cleaved DNA fragment, only a sucrose density gradient centrifugation method, extraction from an electrophoresed gel, or the like can be used to obtain only a fragment having an optimum length.

Suitable vectors are phages or plasmids that can autonomously grow in the host microorganism.

Examples of the phage include Escherichia coli (Es
When cherichia coli) is used as the host microorganism, λ phage and M13 phage can be used.

Further, as the plasmid, for example, Escherichia
When using Escherichia coli as the host microorganism, pUC118, pBR322, pU
C18 and their derivatives can be used. Furthermore, it is also possible to use, for example, a pHY300PLK or yeast vector capable of autonomous growth in two or more types of host microorganisms such as Escherichia coli and Bacillus subtilis. Such a vector is cut with a restriction enzyme or the like in the same manner as the DNA described above to obtain a vector fragment. The method of ligating the DNA fragment and the vector fragment may be a method using a known DNA ligase. For example, after annealing the DNA fragment and the vector fragment, an appropriate DN in vitro is used.
Recombinant DNA is created by the action of A ligase. If necessary, it can be introduced into a host microorganism after annealing and then converted into a recombinant DNA by utilizing in vivo DNA ligase.

Any host microorganism may be used as long as the recombinant DNA is capable of stable and autonomous growth and capable of expressing its trait.

The method for introducing the recombinant DNA into the host microorganism is a known method, for example, the calcium method (Lederberg, EM and Cohen, SN, J. Bacte when the host microorganism is Escherichia coli).
riol., 119 , 1072, (1974)) can be adopted.

For λ phage DNA, in vitro packaging method (Horn, B., Methods in Enzymology, 68 , 299, (197
9)) was used to form λ phage particles, and the λ phage particles were added to the culture suspension of Escherichia coli,
It is possible to obtain a special transduction phage having HWP-producing ability.

The method for selecting the transformed microorganism into which the recombinant DNA has been introduced is the DNA-DNA hybridization method using a gene having a high homology with the target gene as a probe. As the liquid selection medium, a minimal medium or an antibiotic-containing medium is appropriately used depending on the marker on the vector.

Hereinafter, the present invention will be described in more detail and specifically with reference to Examples.

Example Bacillus brevis HPD31 (FERM
BP-1087) After shaking culture at 37 ° C., the cells were collected by centrifugation and washed, and the bacteria obtained were subjected to the method of Saito, Miura (S
aito, H.and Miura, K., Biochim.Biophys.Acta , 72, 619 (1
964)) to separate the chromosomal DNA. Isolated chromosome
The DNA was dissolved in Trisbacher, the restriction enzyme Bam HI (Takara Shuzo Co., Ltd.) was added, and it was partially digested at 37 ° C. Then, the digested product was electrophoresed and the chromosomal DNA fragment was isolated and obtained by extraction from the gel. .

The λ-phage cloning vector EMBL3 was used as the vector. This phage vector EMBL3
EMBL3 DNA obtained by cutting with Bam HI and the above Bacillus
The chromosomal DNA fragment obtained from Brevis HPD31 strain was mixed, and T4DNA ligase (Takara Shuzo) was added for ligation treatment (Weiss, B.Sablon, AJ, Live, TR, Fareed, GCand).
Richardson, CC, J. Biol. Chem., 243, 4543 (1968)).

The treatment liquid thus obtained was added to an in vitro packaging kit (manufactured by STARAT GENE),
In vitro packaging method (Horn, B., Methods in
The DNA was introduced into phage particles by Enzymology, 68,299 (1979) to prepare a DNA library of B. brevis HPD31 strain.

After infecting this with Escherichia coli Q359,
Plaque hybridization was carried out using the MWP gene of Bacillus brevis strain 47 as probe A to obtain positive recombinant DNA φ-SK10.

The MWP gene of Bacillus brevis 47 has already been elucidated by the present inventors (J. Bacteriol., 170, 9
35 (1988)), of which the Hpa I-Hpa I fragment (plasmid pCWP300) was used as probe A (Fig. 3).

A 14 kb BamHI insert fragment was observed in the obtained φ-SK10n, and it was confirmed that this fragment contained only the 5'half of the HWP structural gene (Fig. 4).

Then, the Bg1 II 2.7 kb portion on the 3'side was cloned into Escherichia coli with the plasmid pUC118 by the following treatment to obtain the plasmid pSK31C (5.3 kb) (Fig. 4).

That is, after chromosomal DNA of Bacillus brevis HPD31 was isolated by a conventional method, DNase was digested with the restriction enzyme Bgl II.
After completely digesting A, chromosomal DNA fragments are separated by a conventional method,
I got it.

On the other hand, the plasmid pUC118 used as a vector was treated with the restriction enzyme Bam HI, brought into contact with the DNA fragment obtained above and ligated with ligase.
Using this treatment solution, using the Escherichia coli JM103 strain as a host, the Lederberg, Cohen method (Lederberg, EMand Coh
The plasmid was introduced by em, SN, J. Bacteriol., 119, 1072 (1974) to prepare a transformant.

Regarding the obtained transformants, Cl of φ-SK10 obtained above was used.
Colony hybridization was performed using the aI-Bam HI 400 bp fragment as probe B.

There, positive colonies were screened and the plasmid was isolated therefrom. This plasmid was a novel one which was unknown so far, and was named plasmid pSK31C (Fig. 4). Then, subsequently, the nucleotide sequence of the DNA fragment containing the HWP gene was determined, and the results shown in FIG. 1 were obtained for the amino acid sequence predicted therefrom. The 5'side is shown enlarged in FIG.

In the nucleotide sequence diagram for 4.3 kb from ClaI to BglII shown in FIG. 1, it can be seen that the HWP gene has TTG or ATG as the start codon and TGA as the stop codon.

Therefore, when the N-terminal amino acid sequence of HWP was determined, it was confirmed that it coincided with the 15 underlined amino acids in FIG. Therefore, although the translation start point is unknown,
HWP is synthesized as a precursor protein containing a signal peptide consisting of 53 or 23 amino acids, and its mature form has alanine (position 1) at the N-terminus and valine (position before the stop codon TGA) at the C-terminus. It was fully suggested that the protein has a molecular weight of about 118,000. In other words, the HWP gene, which is the main protein gene, has a GCA (1
Position) to the GTG corresponding to valine.

The amino acid sequence of this HWP contains a large amount of 43% hydrophobic amino acids and 18 amino acid amino acids.
%, Which is contained in a relatively large amount, which is in agreement with the amino acid sequences of surface proteins of various bacteria, but is greatly different from the conventionally known one in that one molecule of cysteine is contained.

Further, as is clear from FIG. 2, this base sequence has two signals (positions at −23 and −53), which is characteristic. And the SD ₁ sequence is found upstream of it.

As a result of investigating the transcription initiation region of the HWP gene by Sl mapping, as shown in FIG. 2, P ₁ ,
Five transcription initiation regions indicated by P ₂ , P ₃ , P ₄ , and P ₅ were confirmed. Since all of these promoters are strong, it is possible to utilize this promoter portion to bind other useful genes and express them strongly. Since the promoter is that of the original bacterium (host), its expression is good and it is very effective.

Further, according to the present invention, the self promoter and main protein gene can be re-introduced, whereby the same gene can be duplicated and the protein production amount can be greatly improved. As a result, the production of food proteins and feed proteins will be greatly increased, and costs will be reduced.

Furthermore, since the entire DNA base sequence is clarified as shown in FIG. 1, it is possible to process and express a useful portion, a fragment of only a desired portion, and thus the present invention can be applied to various kinds of fragments. It can also be used in a wide range of applications.

Two Inverted repeats, which are considered to be terminators, were found downstream of the termination codon TGA in this entire nucleotide sequence, and it is suggested that these terminate transcription of the HWP gene.

(Effects of the Invention) The HWP gene was cloned for the first time by the present invention, and its gene structure was clarified.

Therefore, by expressing this HWP gene, H
WP can be produced in large quantities and can be effectively used as food protein or feed protein.

Further, since the five promoters P ₁ to P ₅ in the gene is a potent both leverages this promoter portion, combining useful genes other can strongly express this. Since the promoter is that of the original bacterium (host), its expression is good and it is very effective.

Further, according to the present invention, the self promoter and main protein gene can be re-introduced, whereby the same gene can be duplicated and the protein production amount can be greatly improved. As a result, the production of food proteins and feed proteins will be greatly increased, and costs will be reduced.

Furthermore, since the entire DNA base sequence is clarified as shown in FIG. 1, it is possible to process and express a useful portion, a fragment of only a desired portion, and thus the present invention can be applied to various kinds of fragments. It can also be used in a wide range of applications.

[Brief description of drawings]

FIG. 1 is a diagram showing the entire base sequence of the gene according to the present invention, and FIG. 2 is an enlarged view of the upstream portion thereof. FIG. 3 is a restriction enzyme map of the main protein DNA of B. brevis 47. FIG. 4 is a drawing showing a restriction enzyme digestion map of the DNA fragment of B. brevis HPD31 (in φ-SK10), a restriction enzyme digestion map of DNA of the main protein of HPD31 strain, and plasmid pSK31C.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:08) (72) Inventor Norihiro Tsukakoshi 2-510 Inotakadai, Meito-ku, Nagoya, Aichi Prefecture (72) ) Inventor Shigezo Utaka 1-24-3 Uenoencho, Meito-ku, Nagoya-shi, Aichi

Claims (4)

[Claims] 1. A DNA having a nucleotide sequence corresponding to an amino acid sequence starting from the +1 position (alanine) in FIG. 1 and ending at the final position (valine). 2. The position (methionine) at -23 in FIG. 1 and / or
Alternatively, a DNA having a base sequence corresponding to an amino acid sequence containing a translation start point at position -53 (methionine) and starting from position +1 (alanine) and ending at the final position (valine). 3. A transcription start point at the position of P ₁ , P ₂ , P ₃ , P ₄ and / or P ₅ in FIG. ₁ and at a position of -23 (methionine) and / or a position of -53. A DNA having a base sequence corresponding to the amino acid sequence including the translation initiation point of (methionine) and further starting from the +1 position (alanine) and ending at the final position (valine). 4. A DNA having the nucleotide sequence shown in FIG. 1 from the first codon ATC to the final codon TCT.