JPH01300889A - Transformant and production of mtgase using same - Google Patents

Abstract

NEW MATERIAL:A microorganism such as Escherichia coli transformed by a plasmid containing gene coding guinea pig liver transglutaminase (MTGase). USE:For production of MTGase to modify nutrient value or physical properties by introducing various amino acids into food proteins so as to form covalent bond. PREPARATION:For example, from clones from plural Escherichia coli containing the partially duplicate CDNA of MTGase, plasmid pLTG 16 and plasmid pLTG 21 are extracted and cut with a relevant restriction enzyme, and the resultant DNA fragments are fractionated by agarose electrophoresis followed by conjunction of the DNA fragments in the translational region coding MTGase using DNA ligase to produce a plasmid containing gene coding MTGase. Thence, this plasmid is incorporated into Escherichia coli to transform this Eschrichia coli followed by culture of the resultant microorganisms, thus obtaining the objective recombinant MTGase.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a microorganism transformed with a plasmid containing a gene encoding MTGase and a method for producing recombinant MTGase using the microorganism.

<Prior art> Guinea pig liver transglucuminase (Protein
-glutamine: amine r -gluta
myltransferase.

EC2, 3, 2, 13 (hereinafter abbreviated as TGase) is one of the protein modification enzymes and is a Ca''-dependent acyltransferase.The substrate is a Gln residue in the peptide chain as an acyl donor. The γ-carboxamide group of
The primary amino group of the amine compound and the ε-amino group of the Lys residue in the peptide chain react as acyl acceptors.

The reaction mechanism is as follows.

+++ vr; == ri=
=0=υ b: (j
(:): (Jl 1
1 t,,IA Mi0=0 In other words, the reaction catalyzed by this enzyme is the reaction between the Gin residue in the peptide chain and the Lys residue in the peptide chain.
These include the formation of T-glutamyl)lysine crosslinks, the introduction of an amine compound to the Gin residue in the peptide chain, or the deamidation reaction of the Gin residue in the peptide chain in the absence of an amine compound.

This TGase has been found in various parts of many animals, but its physiological function is mostly related to the formation of crosslinks between proteins, and crosslinking of fibrin monomers, which is the final step in the blood coagulation cascade reaction. These include stabilization through polymerization, formation of insoluble proteins in the stratum corneum of epidermal tissue, and crosslinking of hair proteins.

TGase can be used to study protein biochemistry, to form new oxygen reaction systems, to form reusable coenzyme derivative-casein complexes, and to improve the nutritional value of food proteins by introducing essential amino acids. It would be desirable to develop a method that can be improved and thus obtained in large quantities.

Now, when using TGase, guinea pig liver TGase (hereinafter abbreviated as MTGase) is currently used as a source of high quality enzyme.

However, although MTGase is qualitatively good,
There are disadvantages such as the amount that can be obtained is limited because the source is guinea pig liver, the purification method is very complicated and the yield is extremely low, and furthermore, the guinea pig itself is very expensive. . Therefore, from before, MTG
It has been desired to provide a method for producing e in large quantities, inexpensively, and more simply.

<Problem to be solved by the present invention> The problem to be solved by the present invention is to produce a large amount of desired recombinant M from microorganisms using genetic engineering techniques.
The present invention provides a method for producing TGase.

<Means for solving the problems of the present invention> As a result of intensive research to solve the above problems, the present inventors found that by culturing a microorganism transformed with a plasmid containing a gene encoding MTGase, , the desired recombinant MTGase could be produced in large quantities, and the present invention was completed.

That is, the present invention is a microorganism transformed with a plasmid containing a gene encoding MTGase, and a method for producing recombinant MTGase using the microorganism.

The invention will be explained in detail below.

DNA sequence encoding MTGase and MTGase
The amino acid sequence of
l, Chem, 51(3).

957-961 Shin (1987)) According to this, MT
The DNA encoding Gase has a start codon (ATG)
It contains a codon consisting of 690 amino acid residues (Fig. 2r).The molecular weight of this enzyme is calculated to be 76.620.Now, regarding the preparation of a microorganism that produces MTGase, first, MTGase is The encoding gene is incorporated into a plasmid that can be replicated within the microorganism.

At this time, the gene encoding MTGase may be inserted downstream of the promoter sequence of the expression vector.

The method for integration is to cut the plasmid with an appropriate restriction enzyme, insert the gene encoding the desired MT[;ase into the cut site, and connect. The recombinant DNA thus obtained can be introduced into a prokaryotic host, and a strain that produces MTGase can be selected from among the transformed microorganisms obtained. In the present invention, Escherichia coli, Bacillus subtilis, etc. can be used as the microbial host into which the recombinant DNA is introduced, but Escherichia coli is preferably used.

Furthermore, various vectors for Escherichia coli that can be used in the present invention include EK type plasmid vectors (relaxed type), EK type plasmid vectors (stringent type), and λgt type phage vectors.

In addition, promoters include trp promoter, Iac
All promoters that function in Escherichia coli are available, including the promoter.

The following methods are commonly used for transforming host cells using recombinant DNA. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up this DNA can be transformed by the well-known CaC1 method after harvesting cells in the logarithmic growth phase. MgCj22 or RbC in the transformation reaction solution
If A coexists, transformation efficiency will be improved. It is also possible to transform host cells after preparing protoplasts.

The culture medium and culture method for culturing the transformed microorganism may be any conventional culture medium or culture method. That is, the culture medium is a conventional medium containing a carbon source, a nitrogen source, inorganic ions, and, if necessary, organic micronutrients such as amino acids and vitamins.

As the carbon source, glucose, sucrose, etc., starch hydrolysates containing these, molasses, etc. are used. Ammonia gas, aqueous ammonia, ammonium salts, and others can be used as the nitrogen source.

More preferably, natural materials such as peptone, tryptone, meat extract, and yeast extract are also used.

Cultivation may be carried out under aerobic conditions while adjusting the pH and temperature of the medium as appropriate.

To collect recombinant MTGase from cultured bacterial cells, the following method may generally be used.

That is, after the cultured cells are collected using a refrigerated centrifuge or the like, they are suspended in an appropriate buffer, and the cells are disrupted using ultrasound or a dyno mill to obtain an extract. This bacterial cell extract is subjected to ammonium sulfate precipitation fractionation method, ion exchange chromatography method, gel filtration method, antibody column method, etc. to produce recombinant MTG
A purified sample of e is obtained.

〔effect〕

Since the present invention uses guinea pig liver as a source, it is an innovative method that can overcome the drawbacks of conventional methods such as: 1. Only a small amount of MTGase can be provided; 2. Expensive; and 2. Very complicated purification procedure. It is. In other words, by using the method of the present invention, MTG gas can be produced in large quantities, cheaply, and easily.
e.

The nutritional value and physical properties of the recombinant MTGase obtained in this manner can be modified by covalently introducing various amino acids into food proteins, similar to conventional natural MTGase. In addition, this enzyme can be expected to be applied to pharmaceuticals and chemical products other than foods.

Hereinafter, the present invention will be explained according to examples.

[Example 1: Construction of expression plasmid pKTG 1] The present inventors have already obtained five partially overlapping cDNA clones of MTGase (Daiichi Kokusei), and based on this, D
Determining the NA sequence and amino acid sequence (second country)
.

Now, first of all, the plasmid pi shown in Fig. 1, TG 16
Escherichia co+JMC1061 (hereinafter referred to as
E.

coli MC1061) and plasmid pLT
Plasmid pLTG16 and plasmid pLTG21 were extracted from E. coli MC1061 containing G21 according to the following method.

That is, 100ml of the culture solution was centrifuged to collect only the bacterial cells, and then mixed with 50mM Tris-HCJ, pH 7.5.
n+j! After freezing at -80°C, thaw and add lysozyme (final concentration, 2 mg/m/m).
Let stand for 10 minutes, then add EDTA (final concentration 0.1
Add M) and let stand at 0°C for 10 minutes. After that, Tri
Add tonX-100 (final concentration 0.1%) and let stand at 0°C for 60 minutes. Then 30. OOOrpm, 30
Centrifuge for a minute and treat the supernatant with an equal volume of water-saturated phenol. The aqueous layer was further treated with an equal volume of chloroform, the aqueous layer was extracted, and the aqueous layer was added to a final concentration of 20 μg/m
RNase was added to the solution at a concentration of / and incubated at 37° C. for 60 minutes.

After that, 0.2 volume of 5M NaCl and 1/3 volume of polyethylene glycol were added, and after standing at 0°C for 60 minutes, 10.0
Collect the DNA precipitate by centrifugation at 0 Orpm for 20 minutes.

Next, add 3.8ml of this precipitate! of C5 dissolved in 4 g of water.
After dissolving by adding C (1), 200 μl of 10 mg/mj! EtBr was added and the mixture was heated at 40,000 rpm for 16 hours.
Perform ultracentrifugation fractionation at 20'C.

After centrifugation, the plasmid DNA fraction was extracted and saturated with water.
- EtB by performing four extractions with 1 to 2 volumes of butanol.
Exclude r. After that, dialysis was performed in nzo and CsCj1!
After removing , 1/10 volume of 3M sodium acetate pH 5,6 is added, and further 2 volumes of cold ethanol are added, and the mixture is allowed to stand overnight at 20°C. Collect this ethanol precipitate by centrifugation and
After washing with 0% ethanol aqueous solution, dry thoroughly, and add 50 μl of this precipitate to 10 mM Tr containing 1 mM EDTA.
is-tlcj! , dissolved in pH 7.5 and used as a sample.

MTGase expression plasmid pKTG 1 was constructed from plasmid pLTG16 and plasmid pLTG21 (Figures 3 and 4). The details are shown below.

i) Construction of plasmid LTG 1621A (a) Now, plasmid pLTG16 was cut with the restriction enzyme Bsm 1 s old ndI [[], and the larger DNA fragment was recovered by agarose gel electrophoresis fractionation. In addition, Figures 3 and 4
In the figure, the DNA fragments used among the DNA fragments cut with restriction enzymes are indicated by broken lines.

(b) Plasmid pLTG21 was cut with restriction enzymes Bsm I and tlindll, and a DNA fragment of about 740 bp was recovered by agarose gel electrophoresis fractionation.

(c) DNA fragments obtained in (a) and (b) above
Ligate using ligase to create plasmid pLTG162.
1A was prepared.

ii) Construction of plasmid LTG 1621B (a) The plasmid pLTG1621A obtained in i) above was cut with the restriction enzymes Sal r and old ndI, and a small DNA fragment (approximately 1440 bp) was separated by agarose electrophoresis.
was recovered.

(b) Plasmid pLTG21 was transformed with the restriction enzyme old ndI [I
and EcoRr, and a small DNA fragment (approximately 740 bp) was recovered by agarose electrophoresis fractionation.

(c) Plasmid pUc9 (manufactured by Pharmacia) was cut with restriction enzymes 5alT and EcoRI, and a large NΔ fragment was recovered by agarose electrophoresis fractionation.

(d) The DNA fragments obtained in (a), (b), and (c) above were ligated with DNA ligase to create plasmid pLTG16.
21B was constructed.

iii) Construction of plasmid UTG 1 (a) ii above
) plasmid pLTG1621B obtained with restriction enzyme N
After cutting with coI and BstEII, a DNA fragment of approximately 370 bp was recovered by agarose electrophoresis fractionation.

(b) Plasmid pLTG1621 obtained in ii) above
B was cut with restriction enzymes BstEn and Sal I, and the smaller DNA fragment (approximately 1
700bp) was recovered.

(c) Plasmid puc 1) BN (kindly provided by Dr. Wills Kenmaki, Kyoto University) was cut with restriction enzymes, Nco I and 5alT, and the larger DNA fragment was recovered by agarose electrophoresis fractionation.

(d) The DNA fragments obtained in (a), (b), and (c) above are ligated using DNA ligase to create plasmid ρUTG.
1 was constructed.

iv) Construction of TGI in a plasmid (a) The plasmid pUTG i obtained in iii) above was cut with restriction enzymes Nco I and BstEII, and a DNA fragment of about a7 obp was recovered by agarose electrophoresis fractionation.

(b) The plasmid purc 1 obtained in iii) above was cut with restriction enzymes BstEII and Ps, and the smaller DNA fragment (approximately 1700
bp) was recovered.

(c) Plasmid pKK233-2 (Pharmacia) having trc promoter (fusion of trp promoter and lac promoter) and ampicillin resistance was cut with restriction enzymes Nco I and Pst I, and fractionated by agarose electrophoresis. Large NA fragments were collected.

(d) The DNA fragments obtained in (a), (b), and (c) above were ligated using DNA ligase to construct MTGase expression plasmid pKTG1.

That is, by performing the above operations, plasmids pLTG16 and pLTG16 having two types of overlapping cDNAs were created.
A plasmid pKTG 1 containing the entire DNA sequence encoding MTGase from G21 (from the start codon ATG to the stop codon TAA) was constructed.

[Example 2 Recombinant MTGase using Escherichia coli]
i) Escherichia co' carrying the plasmid pKTG 1 prepared in Example 1
JJM103 strain (FERM p-10008) at 50
．． cog/m! ! 2YT medium containing ampicillin (1,
6% batato tryptone, 1.0% yeast extract, 1.0%
Na (/!, pH6,7)1. Off at 37℃, 12
Cultured for hours.

Thereafter, 0.2 ml of isopropylthiogalactoside (IPTG) was added as an inducer and cultured for 5 hours.

The cultured bacteria were collected by centrifugation, and 0.15M KCZ
After washing with 20mM Tris-HCl buffer (pH 7.5) containing 2mM EDTA and 0.2mM DTT, it was suspended in the same buffer 30w+1.

A portion of the collected bacterial cells was taken and lysed with 1% SO3, and a portion of the extract was subjected to 5OS-polyacrylamide gel electrophoresis. As a control, MTGase preparation purified from guinea pig liver and plasmid pKK233-
Escherichia coli JM103 strain transformed with 2 was used.

This electrophoresed acrylamide gel was transferred onto a nitrocellulose membrane, reacted with an anti-MTGase antibody, and then reacted with a peroxidase-conjugated secondary antibody. The results of this Western blotting are shown in FIG.

As will be seen, Escherichia coli strain JM103 (FERM P-
10008) produced recombinant MTGase within the bacterial cells.

ii) Since it was determined that MTGase protein was produced within the bacterial cells according to i) above, this recombinant MTGase was extracted from the bacterial cells by the following method. That is, the above suspension was subjected to sonic treatment (20 kilocycles,
300 seconds) and extracted.

This extract showed transglutaminase activity (Figure 6). As a control, plasmid pKK2
Escherichia coli JM103 transformed with 33-2
An extract extracted from the strain in the same manner was used.

As can be seen from Figure 6, MTGase is a Ca2+-dependent enzyme, so in the absence of Ca, even if FER
MP-16oθ? Even the extract of 100% does not show transglutaminase activity.

Note that transglutaminase activity was measured by the following method.

5mg/n*acetyl α1-casein, 0.13mM
[3H] Putrescine, 50mM) Lis-HCl (pH 7,
5).

Reaction 1 (150 μN) containing 5mM CaC12, 10mM DTT, and extract solution is incubated at 37° C., and a fixed amount of the reaction solution (20 μl) is spotted on a paper disk at fixed time intervals. After washing unreacted [3H]putrescine with trichloroacetic acid, the radioactivity incorporated into acetyl α,l-casein fixed on the paper disk was measured using a scintillation counter.

(L, Roland et al.: 8n
a1. Biochem, +50+623-
631 (1972)).

[Example 3 Purification of recombinant MTGase using monoclonal antibody column] 30ml of the extract obtained in Example 2ii) was purified using anti-MTGase.
e It was applied to a monoclonal antibody column (1.4 cm x 6 cm).

TBS buffer (20mM) Lis-HCl (pH 7,
5), 0.15M MC210, 2mM DTT, 2
After washing with 100 mA (mM IEDTA), elution buffer 7 (20mM NaHCO:+-NaOH
(pH 10,4), 0.4mM DTT, 2mM
EDTA.

The target recombinant MTGase was eluted at 2M KCj2>40m+2.

The eluate was mixed with 20 ml of counter buffer (1M Tris-HCl (pH 7.5, 0.4mM DTT, 2mM
Neutralized with EDTA).

Thereafter, purified recombinant MTGase was obtained by ultra-overtreatment.

For confirmation, SOS-PAGE was performed and a uniform band was obtained. This isolated recombinant MTGa
When the transglutaminase activity of se was measured, its specific activity was 1690 units/mg x 10'.

[Brief explanation of the drawing]

FIG. 1 shows the translated region of MTGase CDNA and clones containing each gene region. Figure 2 shows the amino acid sequence of MTGase and MTGase.
The DNA sequence encoding is shown. FIG. 3 shows a diagram of the construction of plasmid pLrc1621B. FIG. 4 shows a diagram of the construction of plasmid pKTG1. Figure 5 shows Escherichia coli strain JM103 (FERM P-
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Claims (3)

[Claims] (1) Guinea pig liver transglutaminase (MT
A microorganism transformed with a plasmid containing a gene encoding Gase). (2) Claim (1) that the microorganism is Escherichia coli
Microorganisms mentioned. (3) Production of recombinant MTGase, which comprises culturing the microorganism according to claim (1) or (2) in a medium to produce the desired recombinant MTGase, and collecting the recombinant MTGase from the medium. Law.