JPH1075787A - Variant type alanine aminotransferase and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new enzyme comprising a variant from which plural amino acids at N end of human alanine aminotransferase are lost, having higher generic engineering productivity than a natural type enzyme and useful in a clinical inspection field as an index for liver function disorders. SOLUTION: This enzyme is a new variant type human alanine aminotransferase (hALT) comprising an amino acid sequence represented by 10th to 495th amino acids of an amino acid sequence represented by the formula, loosing at least 5 amino acids of the N end therefrom in human alanine aminotransferase and having human alanine aminotransferase activity and is capable of producing in large amounts in a microbial host, compared with natural type hALT conventionally having only low productivity and capable of using the blood concentration as an index of disorder of liver function and useful in a field of clinical diagnosis as a standard serum in measuring the concentration. The enzyme is obtained by screening cDNA library derived from human cell to collect natural type hALT gene, applying site-specific variation thereto and expressing the gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to a mutant human alanine aminotransferase in which a genetically modified product is produced in a microbial host with high efficiency and a method for producing the same, which is useful as an enzyme for clinical diagnosis.

[0002]

2. Description of the Related Art Alanine aminotransferase (A)
lanine aminotransferase,
E. FIG. C. 2.6.1.2, hereinafter abbreviated as ALT, is generally GPT (Glutamic pyruvic tr).
The enzyme is widely used in the field of clinical diagnosis because its blood concentration serves as an indicator of impairment of liver function. For use of ALT in clinical diagnosis, a standard enzyme is indispensable, and human-derived ALT (hereinafter abbreviated as hALT) has been produced by purification from biological components or cell culture. However, purification of biological components and production of hALT by cell culture are complicated and very expensive, and for the purpose of solving this problem, a method for producing a recombinant in an Escherichia coli host using a full-length hALT gene has been developed. (JP-A-5-68548 and JP-A-8-103278).

[0003]

However, these hALs
The method for producing a recombinant of T in an E. coli host also cannot be said to have sufficiently improved the production efficiency, and a method for further increasing the productivity has been demanded in order to supply hALT inexpensively and stably. .

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies on the above problems and found that at least 5 amino acids, preferably 9 amino acids (including the amino acid encoded by the initiation codon) at the N-terminal of natural hALT. A) a mutant hALT microbial host gene recombinant represented by 10 to 495 of the amino acid sequence of SEQ ID NO: 1
Enzymatic properties of enzyme activity, Michaelis constant, heat stability and pH stability are almost the same as those of natural hALT, and at least 5 amino acids are deleted compared to the recombinant microorganism host of natural hALT. Mutated hALT
It has been discovered that the mutant hALT is unexpectedly expressed with high efficiency due to its genetic properties and its polypeptide properties, thereby completing the present invention.

That is, the present invention relates to an ALT in which at least 5 amino acids at the N-terminus of natural hALT are deleted.
A mutant hALT having an activity, comprising an amino acid sequence represented by amino acids 10 to 495 of the amino acid sequence of Sequence Listing 1,
A mutant hALT having LT activity, a DNA comprising an amino acid sequence represented by 10 to 495 of the amino acid sequence of Sequence Listing 1 and encoding the amino acid sequence of mutant hALT having ALT activity, exogenous to the host, and naturally occurring A substantially pure recombinant microorganism characterized in that it has the ability to produce a mutant hALT having an ALT activity in which at least 5 amino acids at the N-terminus of the hALT are deleted, from 10 of the amino acid sequence in SEQ ID NO: 1 A substantially pure genetically modified microorganism comprising the amino acid sequence represented by SEQ ID NO: 495 and capable of producing a mutant hALT having ALT activity, represented by 10 to 495 of the amino acid sequence of Sequence Listing 1. The amino acid sequence
It is a substantially pure genetically modified microorganism having DNA encoding the amino acid sequence of mutant hALT having T activity.

[0006] The present invention further provides that the host is exogenous and has at least 5
Mutant hALT having ALT activity lacking amino acids
A method for producing a mutant hALT, which comprises culturing a substantially pure genetically modified microorganism having the ability to produce ALT, comprising an amino acid sequence represented by 10 to 495 of the amino acid sequence of SEQ ID NO: 1; Mutant hALT having
A method for producing a mutant hALT, which comprises culturing a substantially pure genetically modified microorganism having the ability to produce ALT, comprising an amino acid sequence represented by 10 to 495 of the amino acid sequence of SEQ ID NO: 1; Mutant hA having activity
A method for producing a mutant hALT, which comprises culturing a substantially pure recombinant microorganism having a DNA encoding the LT amino acid sequence, wherein at least 5 amino acids at the N-terminus of natural hALT are deleted. A composition for analyzing a mutant hALT having an ALT activity, which is composed of an amino acid sequence represented by amino acids 10 to 495 of the amino acid sequence of Sequence Listing 1 and having a ALT activity.

[0007] In order to produce a mutant hALT in which at least the N-terminal 5 amino acids of natural hALT are deleted,
The following may be performed. That is, a mutant hALT gene obtained by isolation and mutation from a cDNA library or total synthesis of DNA is incorporated into an appropriate vector, introduced into a microorganism to obtain a transformant, and the transformed microorganism is cultured. Extract mutant hALT from
It may be purified.

[0008] There is no particular limitation on the method for obtaining the hALT gene, and the amino acid sequence (Ish) of a known natural hALT from a human cell-derived cDNA library is known.
iguro M .; , Et al. , Biochemis
try, 1991, 30, 10451-10457) and activity expression, and may be isolated by a conventional gene cloning technique, or the entire gene DNA may be synthesized. A human cell-derived cDNA library may be prepared from human biological components by conventional genetic engineering techniques, or a commercially available human cell-derived cDNA library may be used.

[0009] If the natural hALT gene is first isolated, screening may be carried out from a cDNA library by the oligonucleotide probe method based on the known amino acid sequence of the natural hALT,
You may separate by PCR method. Screening may be performed from a cDNA library using ALT activity expression as an index. In addition, all gene DNA can be synthesized.

[0010] There is no particular limitation on the method for preparing a mutant hALT gene from the natural hALT gene thus isolated, and a method conventionally used in gene recombination techniques can be used. For example, hA
Site-directed mutagenesis and PCR using oligonucleotide primers designed and synthesized to have a DNA sequence similar to the 5 'end of the LT structural gene and excluding the DNA encoding the amino acid to be deleted Method, synthesis D
Incorporation of NA into N-type of natural hALT
Mutant hAL having at least 5 amino acids removed from its terminal
The gene for T may be prepared, and the upper limit of amino acid removal from the N-terminus may be any as long as the mutant hALT has good productivity and ALT activity, for example, 20 amino acids from the N-terminus. Removal, preferably removal of 15 amino acids.

If the mutant hALT gene is to be directly obtained, one of the two sets of oligonucleotide primers for PCR is used as a set of 5 ′ of the hALT structural gene.
It has the same DNA sequence as the terminal side and is designed to have a structure excluding the DNA encoding the amino acid to be deleted.
It may be synthesized and synthesized from a cDNA library using the PCR method. In addition, all gene DNA can be synthesized.

In gene synthesis by the PCR method, an appropriate restriction enzyme recognition site is generally added to the end of a synthesized DNA fragment in order to simplify subsequent operations. Method. Mutant hAL
As a vector incorporating the T gene, a vector constructed for gene recombination from a phage or a plasmid capable of autonomously growing in a host microorganism is suitable. As the phage vector, for example, a microorganism belonging to the genus Escherichia can be used. Where λgt · λC, λ
gt · λB or the like can be used. As a plasmid vector, for example, when a microorganism belonging to the genus Escherichia is used as a host microorganism, plasmid pBR322,
pBR325, pACYC184, pUC12, pUC
13, pUC18, pUC19, pUC118, Blu
scriptKS +, pTrc99A, pUB110 and pKH300PLK when Bacillus subtilis is used as a host, and pIJ680 and pIJ70 when using actinomycetes as a host.
2. When yeast is used as a host, especially Saccharomyces cerevisiae, YRp7, pYC1, YEp13 and the like can be used. Particularly, a vector having a structure in which a promoter that functions efficiently when a mutant hALT gene is incorporated is suitable upstream. When Escherichia coli is used as a host microorganism, pUC12, pUC18, pUC19,
pUC118, pUC119, pTV119N, pBl
UsscriptSK +, pTrc99A, etc. match this.

In addition, these vectors contain a mutant hAL
The method for incorporating the T gene is not particularly limited, and a conventionally used method can be used. For example, using a suitable restriction enzyme, the DNA containing the mutant hALT gene and the expression vector are treated, and a DNA fragment and a vector containing the mutant hALT gene are obtained from each, and then ligated using DNA ligase. An expression plasmid is obtained by introducing the plasmid into an appropriate microorganism host and extracting and purifying the plasmid from the transformed microorganism.

There is no particular limitation on the microorganism into which the expression plasmid incorporating the mutant hALT gene is introduced, as long as the recombinant DNA can stably and autonomously grow. For example, when the host microorganism is a microorganism belonging to the genus Escherichia, Escherichia coli DH1, Escherichia coli HB101, Escherichia coli W3110, Escherichia coli MV1184 and the like can be used. When the microorganism host is a microorganism belonging to Bacillus subtilis, for example, Bacillus subtilis ISW1214, or a microorganism belonging to actinomycetes, Streptomyces lividans TK2
In the case of microorganisms belonging to Saccharomyces cerevisiae such as Saccharomyces cerevisiae 4, Saccharomyces cerevisiae INVSC1
Etc. can be used.

As a method for transferring the recombinant DNA to the host microorganism, for example, when the host microorganism is a microorganism belonging to Escherichia coli, Saccharomyces cerevisiae, or Streptomyces lividans, the host strain is transformed into a competent cell according to a conventional method. Transfer of the recombinant DNA may be performed, and depending on the strain, electroporation may be used. The presence or absence of the transfer of the target plasmid into the host microorganism may be selected by a selective culture method using a selective medium, based on a drug resistance marker or an auxotrophic marker of the vector.

The method for producing a mutant hALT by culturing a genetically modified microorganism into which a mutant hALT gene has been introduced is not particularly limited, but the method may be carried out in consideration of the nutritional and physiological properties of the transformant microorganism. You only have to choose the conditions,
Usually, in many cases, liquid culture is performed. However, industrially, it is advantageous to perform deep aeration stirring culture. As the nutrient source of the medium, those commonly used for culturing microorganisms can be widely used.

The carbon source may be any carbon compound that can be assimilated by the host microorganism, such as glucose, saccharose, lactose, maltose, fructose, molasses and the like. Any available nitrogen compound may be used as the nitrogen source. For example, peptone, meat extract, yeast extract, casein hydrolyzate and the like are used. In addition, phosphate, carbonate, sulfate, magnesium, calcium,
Salts such as potassium, iron, manganese and zinc, specific amino acids and specific vitamins are used as needed.

The culture temperature is determined by the growth of the host microorganism,
Although it can be appropriately changed within the range for producing ALT, for example, when the host microorganism belongs to Escherichia coli, it is preferably 20 to 42 ° C.
It is about. Culture conditions vary slightly depending on the conditions,
The culture may be terminated at an appropriate time in consideration of the time when the mutant hALT reaches the maximum termination. For example, when the host microorganism belongs to Escherichia coli, it is usually about 12 to 48 hours. The pH of the medium can be appropriately changed within a range in which the host microorganism grows and produces mutant hALT. For example, when the host microorganism belongs to Escherichia coli, the pH is preferably about 6.0 to 8.0.

The method for extracting the produced mutant hALT is not particularly limited, and a conventionally used method can be used. As a specific example of the extraction method, a microbial cell is collected from the obtained culture by means such as filtration or centrifugation, and then the cell is destroyed by a mechanical method or an enzymatic method such as lysozyme and mutated. What is necessary is just to obtain a cell extract containing the recombinant microorganism host of the type hALT.

The recombinant host microorganism of the natural hALT can also be obtained in the same manner as the mutant hALT. Mutant hALT obtained by the above method
The properties of the recombinant microorganism host and the natural hALT derived from human cells are compared as follows. (1) Enzyme action Both natural hALT and mutant hALT catalyze a reaction for producing pyruvic acid and L-glutamic acid from L-alanine and 2-oxoglutarate, or catalyze a reversible reaction thereof, as described below. I do.

[0021]

Embedded image

(2) Molecular weight (calculated from amino acid sequence) Natural hALT: 54500 (495 amino acids) Mutant hALT: 53600 (at 486 amino acids) (3) Michaelis constant Natural hALT: 15.6 mM (substrate: D, L) -Alanine) 0.15 mM (substrate: α-ketoglutarate) Mutant hALT: 15.4 mM (substrate: D, L-alanine) 0.17 mM (substrate: α-ketoglutarate) (4) Thermal stability natural hALT : 100% activity at 45 ° C. for 15 minutes and 92% activity at 50 ° C. for 15 minutes. Mutant hALT: 100% after treatment at 45 ° C. for 15 minutes, and 87% activity after treatment at 50 ° C. for 15 minutes. (5) pH stability Natural hALT: stable between pH 6.0 and 8.0. Mutant hALT: stable between pH 6.0 and 8.0. (6) Productivity of Genetically Recombinant in Microbial Host Natural hALT: mutant hALT = 1: 2 We have found that the mutant hALT microbial host gene recombinant obtained in this way can be used as a natural hALT. In comparison, it was confirmed that there was almost no change in the enzymatic properties and that the productivity of the recombinant microorganism host was high.

[0023]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. In the examples, the operation described as being in accordance with a conventional method is described in, for example, the method of Maniatis et al. (T.m.
aniatis. , Et al. Molecular
Cloning Second Edition.
Cold Spring Harbor Labora
tory 1989) or various commercially available enzymes and procedures attached to kits. Also,
Recombinant DNA experimental enzyme reagents (restriction enzymes, etc.), vector DNA, and kits used in the experiments were purchased from Takara Shuzo Co., Ltd. unless otherwise specified.

[0024]

Example 1 <Acquisition of Natural and Mutant hALT Genes> Amino acid sequences of known natural hALT genes in order to separate natural and mutant hALT genes by PCR and to produce hALT in a microbial host Based on the above, the following three types of oligonucleotide primers were designed. 1) A native hALT in which a restriction enzyme NcoI cleavage site CCATGG is added immediately above the start codon of a native hALT structural gene
Oligonucleotide primer AL for ALT gene synthesis
T1. 2) 27 bases after the start codon ATG (not including the start codon ATG) of the native hALT structural gene are deleted, and the restriction enzyme NcoICC is located immediately above the start codon ATG.
An oligonucleotide primer ALT2 for synthesizing a mutant hALT gene to which an ATGG cleavage site is added. 3) Restriction enzyme Hin downstream of the stop codon of hALT gene
Oligonucleotide primer ALT3, which adds a dIII cleavage site AAGCTT.

The base sequence structure of ALT1 is shown in Sequence Listing 2,
The nucleotide sequence of LT2 is shown in Sequence Listing 3, and the nucleotide sequence structure of ALT3 is shown in Sequence Listing 4. A synthesis request was made for these oligonucleotides (BEX), and a human liver cDNA library (Clontech: QUICK-Clonec).
Using DNA Liver as a template, DNA Therm
al Cycler (Perkin-Elmer-Ce
tus) and a kit for PCR reaction (Takara Shuzo: TaKa)
Ra Taq) and ALT1 and ALT3 for the synthesis of native hALT gene, and ALT2 and ALT3 for the synthesis of mutant hALT gene as primers, PCR was performed according to the manual, and NcoI cleavage site immediately above the start codon. With Hind downstream of the codon
Native and mutant hAL with added III cleavage site
Approximately 1.5 kb DNA fragment containing the T gene was synthesized. The structural gene base sequence (not including start and stop codons) and amino acid sequence of native hALT are shown in Sequence Listing 1.

[0026]

Example 2 <Preparation of Native and Mutant hALT Expression Plasmids>
100 ng of the natural hALT gene-containing DNA fragment synthesized in Example 1 was ligated with 1 u of NcoI and 1 u, respectively.
And a 1.5 kb DNA fragment containing each hALT gene was isolated. This DNA fragment was digested with 1 u of each of the restriction enzymes NcoI and HindIII and dephosphorylated at the cut ends with 1 u of alkaline phosphatase.
By ligating with ligase, plasmid pcALT1 was prepared. This pcALT1 was introduced into an Escherichia coli DH1 strain (ATCC 33849) by a competent cell method, and a transformant Escherichia coli DH1 pcAL was obtained.
T1 was obtained, and pcALT1 was extracted and purified according to a conventional method.

Similarly, a mutant hALT gene-containing D
From the NA fragment and pTV119N, the expression plasmid pcALT2 and the transformant Escherichia coli DH1.
pcALT2 (FERM BP-5615) was prepared. Also, instead of pTV119N, the plasmid pTr
Similarly, pTrcALT1 and a transformant Escherichia coli DH were converted from a native hALT gene-containing DNA fragment using c99A (manufactured by Pharmacia).
1. pTrcALT1 was replaced with a mutant hALT gene-containing D
From the NA fragment, pTrcALT2 and a transformant Escherichia coli DH1 / pTrcALT2 were prepared. FIG. 1 shows pcALT1, pcALT2, pTrcAL.
The structures of T1 and pTrcALT2 are shown.

[0028]

Example 3 <Expression of Mutant hALT> The transformants Escherichia coli DH1, pcALT1, DH1.
pcALT2, DH1 · pTrcALT1, DH1 · p
TrcALT2 was treated with 3.7% BHI (DIF) containing 50 μg / ml ampicillin and 100 μM IPTG, respectively.
(Manufactured by CO, Inc.) overnight at 37 ° C.
After centrifugation at 1,000 G for 1 minute, the cells were collected. Each of these cells was treated with 100 mM Tris-HCl buffer (pH 7.3).
Sonication in the medium, and the cell lysate
An ALT recombinant solution was used.

[0029]

Example 4 <Comparison of Productivity of Genetically Recombinant between Natural ALT and Mutant ALT> A of the natural hALT recombinant solution and the mutant hALT recombinant solution prepared in Example 3
LT activity was measured by the following activity measurement method, and the respective productivity was compared. First reagent 0.5 M Tris-HCl (pH 8.0) 0.16 ml 10 mM NAD 0.10 ml 0.25% nitrotetrazolium blue 0.10 ml 100 u / ml diaphorase 0.05 ml 150 mM α-ketoglutaric acid 0.02 ml 2% Triton X-100 0.10 ml 30 mM ADP 0.10 ml 0.1 M EDTA 0.02 ml 1200 u / ml Glutamate dehydrogenase 0.10 ml Second reagent 1.5 M D, L-alanine 0.25 ml First reagent 0.75 ml and second reagent After mixing 0.25 ml and preheating at 37 ° C. for 3 minutes, an ALT solution 0.02 m
was added and the enzyme reaction was allowed to proceed at 37 ° C. for 10 minutes.
After stopping the reaction by adding 2 ml of% SDS, 550 n
m was measured.

Natural hALT measured by the above method
Table 1 shows the expression efficiencies of the natural hALT gene recombinant and the mutant hALT gene recombinant, and the mutant hALT gene recombinant.

[0031]

Example 5 <Properties of mutant ALT> Natural hALT and mutant h
The enzymatic properties of the ALT recombinants were measured and compared as follows. The mutant hALT recombinants include Escherichia coli DH1 pcA
The one produced by LT2 was used. -Michaelis constant: Km value 0 mM, 120 mM, 240 m of substrate D, L-alanine
M, 360 mM, and 600 mM solutions were prepared, and ALT activity was measured by the above method, and then the Km value was calculated. In addition, 0 mM, 7.5 mM, 1 mM
Prepare 5 mM, 75 mM, and 150 mM concentrations of each solution,
The Km value was calculated in the same manner. -Thermal stability In ice (0 ° C), at 40 ° C, 45 ° C, 50 ° C, 55 ° C,
After keeping the ALT solution containing 0.1% BSA for 15 minutes,
The residual activity value was measured by the above method. PH stability 0.5 M citrate buffer pH 3.0 and 4.0, 0.5
M 3,3 dimethylglutamate buffer pH 4.0,
5.0 and 6.0, 0.5 M phosphate buffer pH 6.0.
0, 7.0 and 8.0, 0.5 M Tris-HCl buffer pH 6.0, 7.0, 8.0 and 9.0, 0.5 M
AL dissolved in glycine buffer pH 9.0 and 10.0
After keeping the T solution (1% BSA added) at 60 ° C. for 5 minutes, the residual activity value was measured by the above method.

Natural hALT measured by the above method
Table 2 shows the Michaelis constant, heat stability, and pH stability of the mutant hALT gene recombinant.

[0033]

[Table 1]

[0034]

[Table 2]

[0035]

EFFECT OF THE INVENTION The mutant hALT of the present invention is a natural hA
Has the same enzymological properties as LT, and has the property that a recombinant can be efficiently produced in a microbial host;
LT production has become easier.

[0036]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1485 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA Origin Organism name: Human Sequence characteristics PCDS sequence GCC TCG AGC ACA GGT GAC CGG AGC CAG GCG GTG AGG CAT GGA CTG AGG 48 Ala Ser Ser Thr Gly Asp Arg Ser Gln Ala Val Arg His Gly Leu Arg 1 5 10 15 GCG AAG GTG CTG ACG CTG GAC GGC ATG AAC CCG CGT GTG CGG AGA GTG 96 Ala Lys Val Leu Thr Leu Asp Gly Met Asn Pro Arg Val Arg Arg Val 20 25 30 GAG TAC GCA GTG CGT GGC CCC ATA GTG CAG CGA GCC TTG GAG CTG GAG 144 Glu Tyr Ala Val Arg Gly Pro Ile Val Gln Arg Ala Leu Glu Leu Glu 35 40 45 CAG GAG CTG CGC CAG GGT GTG AAG AAG CCT TTC ACC GAG GTC ATC CGT 192 Gln Glu Leu Arg Gln Gly Val Lys Lys Pro Phe Thr Glu Val Ile Arg 50 55 60 GCC AAC ATC GGG GAC GCA CAG GCT ATG GGG CAG AGG CCC ATC ACC TTC 240 Ala Asn Ile Gly Asp Ala Gln Ala Met Gly Gln Arg Pro Ile Thr Phe 65 70 75 80 CTG CGC CAG GTC TTG GCC CTC TGT GTT AAC CCT GAT CTT CTG AGC AGC 288 Leu Arg Gln Val Leu Ala Leu Cys Val Asn Pro Asp Leu Leu Ser Ser 85 90 95 CCC AAC TTC CCT GAC GAT GCC AAG AAA AGG GCG GAG CGC ATC TTG CAG 336 Pro Asn Phe Pro Asp Asp Ala Lys Lys Arg Ala Glu Arg Ile Leu Gln 100 105 110 GCG TGT GGG GGC CAC AGT CTG GGG GCC TAC AGC GTC AGC TCC GGC ATC 384 Ala Cys Gly Gly His Ser Leu Gly Ala Tyr Ser Val Ser Ser Gly Ile 115 120 125 CAG CTG ATC CGG GAG GAC GTG GCG CGG TAC ATT GAG AGG CGT GAC GGA 432 Gln Leu Ile Arg Glu Asp Val Ala Arg Tyr Ile Glu Arg Arg Asp Gly 130 135 140 GGC ATC CCT GCG GAC CCC AAC AAC GTC TTC CTG TCC ACA GGG GCC AGC 480 Gly Ile Pro Ala Asp Pro Asn Asn Val Phe Leu Ser Thr Gly Ala Ser 145 150 155 160 GAT GCC ATC GTG ACG GTG CTG AAG CTG CTG GTG GCC GGC GAG GGC CAC 528 Asp Ala Ile Val Thr Val Leu Lys Leu Leu Val Ala Gly Glu Gly His 165 170 175 ACA CGC ACG GGT GTG CTC ATC CCC ATC CCC CAG TAC CCA CTC TAC TCG 576 Thr Arg Thr Gly Val Leu Ile Pro Ile Pro Gln Tyr Pro Leu Tyr Ser 180 185 190 GCC ACG CTG GCA GAG CTG GGC GCA GTG CAG GTG GAT TAC TAC CTG GAC 624 Ala Thr Leu Ala Glu Leu Gly Ala Val Gln Val Asp Tyr Tyr Leu Asp 195 200 205 GAG GAG CGT GCC TGG GCG CTG GAC GTG GCC GAG CTT CAC CGT GCA CTG 672 Glu Glu Arg Ala Trp Ala Leu Asp Val Ala Glu Leu His Arg Ala Leu 210 215 220 GGC CAG GCG CGT GAC CAC TGC CGC CCT CGT GCG CTC TGT GTC ATC AAC 720 Gly Gln Ala Arg Asp His Cys Arg Pro Arg Ala Leu Cys Val Ile Asn 225 230 235 240 CCT GGC AAC CCC ACC GGG CAG GTG CAG ACC CGC GAG TGC ATC GAG GCC 768 Pro Gly Asn Pro Thr Gly Gln Val Gln Thr Arg Glu Cys Ile Glu Ala 245 250 255 GTG ATC CGC TTC GCC TTC GAA GAG CGG CTC TTT CTG CTG GCG GAC GAG 816 Val Ile Arg Phe Ala Phe Glu Glu Arg Leu Phe Leu Leu Ala Asp Glu 260 265 270 GTG TAC CAG GAC AAC GTG TAC GCC GCG GGT TCG CAG TTC CAC TCA TTC 864 Val Tyr Gln Asp Asn Val Tyr Ala Ala Gly Ser Gln Phe His Ser Phe 275 280 285 AAG AAG GTG CTC ATG GAG ATG GGG CCG CCC TAC GCC GGG CAG CAG GAG 912 Lys Lys Val Leu Met Glu Met Gly Pro Pro Tyr Ala Gly Gln Gln Glu 290 295 300 CTT GCC TCC TTC CAC TCC ACC TCC AAG GGC TAC ATG GGC GAG TGC GGG 960 Leu Ala Ser Phe His Ser Thr Ser Lys Gly Tyr Met Gly Glu Cys Gly 305 310 315 320 TTC CGC GGC GGC TAT GTG GAG GTG GTG AAC ATG GAC GCT GCA GTG CAG 1008 Phe Arg Gly Gly Tyr Val Glu Val Val Asn Met Asp Ala Ala Val Gln 325 330 335 CAG CAG ATG CTG AAG CTG ATG AGT GTG CGG CTG TGC CCG CCG GTG CCA 1056 Gln Gln Met Leu Lys Leu Met Ser Val Arg Leu Cys Pro Pro Val Pro 340 345 345 350 GGA CAG GCC CTG CTG GAC CTG GTG GTC AGC CCG CCC GCG CCC ACC GAC 1104 Gly Gln Ala Leu Leu Asp Leu Val Val Ser Pro Pro Ala Pro Thr Asp 355 360 365 CCC TCC TTT GCG CAG TTC CAG GCT GAG AAG CAG GCA GTG CTG GCA GAG 1152 Pro Ser Phe Ala Gln Phe Gln Ala Glu Lys Gln Ala Val Leu Ala Glu 370 375 380 CTG GCG GCC AAG GCC AAG CTC ACC GAG CAG GTC TTC AAT GAG GCT CCT 1200 Leu Ala Ala Lys Ala Lys Leu Thr Glu Gln Val Phe Asn Glu Ala Pro 385 390 395 400 GGC ATC AGC TGC AAC CCA GTG CAG GGC GCC ATG TAC TCC TTC CCG CGC 1248 Gly Ile Ser Cys Asn Pro Val Gln Gly Ala Met Tyr Ser Phe Pro Arg 405 410 415 GTG CAG CTG CCC CCG CGG GCG GTG GAG CGC GCT CAG GAG CTG GGC CTG 1296 Val Gln Leu Pro Pro Arg Ala Val Glu Arg Ala Gln Glu Leu Gly Leu 420 425 430 GCC CCC GAT ATG TTC TTC TGC CTG CGC CTC CTG GAG GAG ACC GGC ATC 1344 Ala Pro Asp Met Phe Phe Cys Leu Arg Leu Leu Glu Glu Thr Gly Ile 435 440 445 TGC GTG GTG CCA GGG AGC GGC TTT GGG CAG CGG GAA GGC ACC TAC CAC 1392 Cys Val Val Pro Gly Ser Gly Phe Gly Gln Arg Glu Gly Thr Tyr His 450 455 460 TTC CGG ATG ACC ATT CTG CCC CCC TTG GAG AAA CTG CGG CTG CTG CTG 1440 Phe Arg Met Thr Ile Leu Pro Pro Leu Glu Lys Leu Arg Leu Leu Leu 465 470 475 475 480 GAG AAG CTG AGC AGG TTC CAT GCC AAG TTC ACC CTC GAG TAC TCC 1485 Glu Lys Leu Ser Arg Phe His Ala Lys Phe Thr Leu Glu Tyr Ser 485 490 495

[0037]

[Sequence list]

SEQ ID NO: 2 Sequence length: 25 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Other nucleic acid
(Synthetic DNA) Sequence CCGGCCATGG CCTCGAGCAC AGGTG 25

[0038]

[Sequence list]

SEQ ID NO: 3 Sequence length: 29 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Other nucleic acid
(Synthetic DNA) Sequence CCGGCCATGG CGGTGAGGCA TGGACTGAG 29

[0039]

[Sequence list]

SEQ ID NO: 4 Sequence length: 27 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Other nucleic acid
(Synthetic DNA) Sequence GGCCAAGCTT CAGGAGTACT CGAGGGT 27

[Brief description of the drawings]

FIG. 1 shows plasmids pcALT1 and pcALT.
2 shows a restriction map of pTrcALT1 and pTrcALT2. "Vector" in the figure is pcALT1, pc
In ALT2, pTV119N, pTrcALT1, pTrcALT
TrcALT2 represents pTrc99A. "HALT gene" in the figure is pcALT1, pTrcAL
In T1, the native hALT structural gene was replaced with pcALT2,
pTrcALT2 represents a mutant hALT structural gene.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication (C12N 1/21 C12R 1:19) (C12N 9/10 C12R 1:19)

Claims (18)

[Claims] 1. A mutant human alanine aminotransferase having alanine aminotransferase activity in which at least 5 amino acids at the N-terminus of human alanine aminotransferase are deleted. 2. The amino acid sequence of SEQ ID NO: 1 from 10 to 49
A mutant human alanine aminotransferase having an alanine aminotransferase activity, comprising the amino acid sequence represented by No. 5. 3. The amino acid sequence of SEQ ID NO: 1 from 10 to 49
5 comprising the amino acid sequence of SEQ ID NO: 5 and encoding the amino acid sequence of a mutant human alanine aminotransferase having alanine aminotransferase activity.
A. 4. The DNA according to claim 3, wherein the DNA is a nucleotide sequence represented by 28 to 1485 of the nucleotide sequence in Sequence Listing 1. 5. A human alanine aminotransferase which is exogenous to a host and has the ability to produce a mutant human alanine aminotransferase having an alanine aminotransferase activity in which at least 5 amino acids at the N-terminal of human alanine aminotransferase are deleted. A substantially pure genetically modified microorganism. 6. The amino acid sequence of SEQ ID NO: 1 from 10 to 49
5. A substantially pure genetically modified microorganism comprising an amino acid sequence represented by No. 5 and capable of producing a mutant human alanine aminotransferase having an alanine aminotransferase activity. 7. The amino acid sequence of SEQ ID NO: 1 from 10 to 49
5 comprising the amino acid sequence of SEQ ID NO: 5 and encoding the amino acid sequence of a mutant human alanine aminotransferase having alanine aminotransferase activity.
A substantially pure genetically modified microorganism carrying A. 8. The substantially pure genetically modified microorganism according to claim 7, wherein the DNA is a nucleotide sequence represented by 28 to 1485 of the nucleotide sequence in Sequence Listing 1. 9. A substantially pure genetically modified microorganism comprising:
6. The substantially pure genetically modified microorganism according to claim 5, which is a microorganism belonging to the genus Escherichia. 10. The microorganism belonging to the genus Escherichia is Escherichia coli DH1 pcALT2 (FERM B).
The substantially pure genetically modified microorganism according to claim 9, which is P-5615). 11. A substantially pure human alanine aminotransferase which is exogenous to a host and has the ability to produce a mutant human alanine aminotransferase having an alanine aminotransferase activity in which at least 5 amino acids at the N-terminal of the human alanine aminotransferase are deleted. A method for producing a mutant human alanine aminotransferase, which comprises culturing a genetically modified microorganism. 12. The amino acid sequence of SEQ ID NO: 1 from 10 to 4
A mutant human alanine aminotransferase comprising an amino acid sequence represented by No. 95 and culturing a substantially pure recombinant microorganism having an ability to produce a mutant human alanine aminotransferase having an alanine aminotransferase activity. Manufacturing method. 13. The amino acid sequence of SEQ ID NO: 1 from 10 to 4
D which encodes the amino acid sequence of a mutant human alanine aminotransferase having an alanine aminotransferase activity
A method for producing a mutant human alanine aminotransferase, which comprises culturing a substantially pure genetically modified microorganism having NA. 14. The DNA has a nucleotide sequence of 28 in the nucleotide sequence of Sequence Listing 1.
14. The method for producing a mutant human alanine aminotransferase according to claim 13, which is a nucleotide sequence represented by SEQ ID NOs. 15. The substantially pure genetically modified microorganism is a microorganism belonging to the genus Escherichia.
A method for producing the mutant human alanine aminotransferase according to the above. 16. The microorganism belonging to the genus Escherichia is Escherichia coli DH1 pcALT2 (FERM B).
The method for producing a mutant human alanine aminotransferase according to claim 15, which is P-5615). 17. A composition for analyzing a mutant human alanine aminotransferase having an activity of alanine aminotransferase, wherein at least 5 amino acids at the N-terminal of the human alanine aminotransferase are deleted. 18. The amino acid sequence of SEQ ID NO: 1 from 10 to 4
A composition for analysis of a mutant human alanine aminotransferase having an amino acid sequence represented by 95 and having alanine aminotransferase activity.