CN1166779C - High-temperature-resistant isocitrate dehydrogenase gene, polypeptide coded by same and preparation method of polypeptide - Google Patents
High-temperature-resistant isocitrate dehydrogenase gene, polypeptide coded by same and preparation method of polypeptide Download PDFInfo
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Abstract
The invention discloses a high-temperature-resistant isocitrate dehydrogenase gene, a polypeptide coded by the gene and a preparation method of the gene. It relates to coding separated DNA with activity or its functional equivalent variant and utilizing recombinant DNA technology to produce polypeptide with high-temp. resistant isocitrate dehydrogenase activity or its functional equivalent variant with the described separated DNA. Based on sequencing and analysis of Tengchong thermophilic anaerobic bacteria whole genome, a high temperature resistant isocitrate dehydrogenase gene is cloned and separated. The gene is useful for preparing transgenic microorganisms or animals and plants for producing a high-temperature-resistant isocitrate dehydrogenase and recovering the enzyme encoded by the gene. In addition, the invention also provides an amino acid sequence and a functional equivalent of another polypeptide with the high-temperature resistance isocitrate dehydrogenase activity. Meanwhile, the invention also provides a method for preparing, separating and purifying the polypeptide with the high-temperature-resistant isocitrate dehydrogenase activity.
Description
Technical Field
The invention relates to mutation or genetic engineering, in particular to a high-temperature-resistant isocitrate dehydrogenase gene, a polypeptide coded by the gene and a preparation method of the gene.
Background
The reaction catalyzed by isocitrate dehydrogenase is as follows:
in the tricarboxylic acid cycle, the isocitrate dehydrogenase catalyzes a reaction that produces 90% of the NACPH available for biosynthesis, thereby producing a large amount of acetate. In bacteria, however, NAD-dependent isocitrate dehydrogenase cannot produce acetate, both lacking the respiratory chain and lacking the full tricarboxylic acid cycle. The isocitrate dehydrogenase of E.coli lacks the Rossmann fold possessed by other dehydrogenases.
Since the three-dimensional structure of isocitrate dehydrogenase and its kinetic metabolic mechanism are known and the structure of its intermediate product has been observed by X-ray crystallization, experimental studies have been made to change metabolic pathways by making minor changes in its structure, and it has been confirmed that the minor changes in structure can cause great changes in catalytic products.
Tengchong thermophilic anaerobe (Thermoanaerobacter tangcongensis) is a microorganism living in hot spring of Tengchong county in Yunnan province of China, is a thermophilic eubacterium (eubacteriia), has the optimal growth temperature of 75 ℃, and grows anaerobically, and gram staining reaction is negative. It was first discovered by the Chinese academy of sciences microorganism and was subjected to taxonomic analysis. The strain is preserved in China center for microbiological preservation MB4T(＝Chinese collection of microorganisms AS 1.2430T＝JCM 11007T). The thermophilic anaerobic bacteria is a species peculiar to China, and high-temperature resistant glutamic imine methyltransferase in vivo also has a peculiar structure.
Disclosure of Invention
It is an object of the present invention to provide an isolated nucleotide sequence encoding a polypeptide having the activity of a thermostable isocitrate dehydrogenase.
Anotherobjective of the invention is to provide an isolated polypeptide with high temperature-resistant isocitrate dehydrogenase activity.
The invention also provides an isocitrate dehydrogenase recombinant vector of thermophilic anaerobe, a host cell containing the recombinant vector and a method for preparing protein.
In one aspect, the present invention provides a nucleotide sequence encoding a polypeptide having the activity of high temperature resistant isocitrate dehydrogenase. Said nucleotide sequence encodes a polypeptide having the amino acid sequence shown in SEQ ID NO.2 or a modified form of said polypeptide which is functionally equivalent or related to another isocitrate dehydrogenase. The nucleotide sequence has a polynucleotide sequence of SEQ ID NO.1 and its mutant forms, the mutant forms include: deletion, nonsense, insertion, missense.
In another aspect, the invention provides a polypeptide with high temperature resistance and isocitrate dehydrogenase activity. The polypeptide has the amino acid sequence in SEQ ID No.2, or conservative variant polypeptide thereof, or active fragment thereof, or active derivative thereof.
The method for producing the high-temperature-resistant isocitrate dehydrogenase comprises the following steps:
1) separating out a nucleotide sequence SEQ ID NO.1 for coding high-temperature-resistant isocitrate dehydrogenase;
2) constructing an expression vector containing a nucleotide sequence of SEQ ID NO. 1;
3) transferring the expression vector in the step 2) into a host cell to form a recombinant cell capable of producing high-temperature-resistant isocitrate dehydrogenase;
4) culturing the recombinant cells in step 3);
5) separating and purifying to obtain the high-temperature-resistant isocitrate dehydrogenase.
The invention relates to separation and expression of a high-temperature-resistant isocitrate dehydrogenase gene of thermophilic anaerobic bacteria. Based on sequencing and analysis of Tengchong thermophilic anaerobic bacteria whole genome, a high temperature resistant isocitrate dehydrogenase gene is cloned and separated. The gene is useful for preparing transgenic microorganisms or animals and plants for producing a high-temperature-resistant isocitrate dehydrogenase and recovering the enzyme encoded by the gene. In addition, the invention also provides an amino acid sequence and functional equivalents of the polypeptide with the high-temperature-resistant isocitrate dehydrogenase activity. Meanwhile, the invention also provides a method for preparing, separating and purifying the polypeptide with the high-temperature-resistant isocitrate dehydrogenase activity.
Drawings
FIG. 1 is a flow chart of the sequencing library construction steps;
FIG. 2 is a flow chart of sequencing and data analysis.
Detailed Description
The invention provides an isolated polynucleotide molecule encoding a polypeptide having the activity of a thermostable isocitrate dehydrogenase, which is obtained by sequencing and analyzing the entire genome of Thermoanaerobacter tengcongensis, has the nucleotide sequence of SEQ ID No.1, encodes a polypeptide having 403 amino acids, which has a predicted molecular weight of 46061 daltons.
The invention also provides a recombinant vector comprising the isolated nucleotide molecule of the invention, and a hostcell comprising the recombinant vector. Meanwhile, the invention comprises a method for constructing the recombinant vector and the host cell and a method for producing the high-temperature-resistant isocitrate dehydrogenase by using a recombinant engineering technology.
The present invention further provides an isolated thermostable isocitrate dehydrogenase or polypeptide having the amino acid sequence of seq id No.2, or at least 70% similar, more preferably at least 90%, 95%, 99% identical.
In the present invention, "isolated" NDA means that the DNA or fragment has been isolated from the sequences which flank it in its natural state, and that the DNA or fragment has been separated from components which accompany the nucleic acid in its natural state, and from the proteins which accompany it in the cell.
In the present invention, "a high temperature-resistant isocitrate dehydrogenase gene" refers to a nucleotide sequence encoding a polypeptide having a high temperature-resistant isocitrate dehydrogenase activity, such as the nucleotide sequence of SEQ.ID No.1 and degenerate sequence thereof. The degenerate sequence is a sequence in which one or more codons in the sequence are replaced by degenerate codons which encode the same amino acid. Due to the known degeneracy of codons, degenerate sequences with homology as low as about 70% to the nucleotide sequence of SEQ ID NO.1 can also encode the amino acid sequence depicted in SEQ ID NO. 2. The term also includes nucleotide sequences that hybridize to the nucleotide sequence of SEQ ID No.1 under moderately stringent conditions, and more preferably, under highly stringent conditions. The term also includes nucleotide sequences having at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% homology to the nucleotide sequence of SEQ ID No. 1.
In the present invention, a polypeptide of an "isolated" protein means that it represents at least 20%, preferably at least 50%, more preferably at least 80%, and most preferably at least 90% (by dry weight or wet weight) of the total material of a sample. Purity can be measured by any suitable method, such as column chromatography, PAGE, or HPLC. The isolated polypeptide is substantially free of components that accompany it in its native state.
In the present invention, "a thermostable isocitrate dehydrogenase" refers to a polypeptide having a sequence of SEQ ID NO.2 having a thermostable isocitrate dehydrogenase activity. The term also includes variants of the sequence of SEQ ID NO.2 which have the same function as a naturally occurring thermostable isocitrate dehydrogenase, including, but not limited to, deletions, insertions and/or substitutions of several amino acids, and additions of one or several amino acids to the C-terminus and/or N-terminus, and also differences in modified forms which do not affect the sequence. For example, it is well known in the art that substitution with amino acids having similar or analogous properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids to the C-terminal and/or N-terminal end does not generally alter the function of the protein. The term also includes active fragments and active derivatives of a thermostable isocitrate dehydrogenase.
In the present invention, various vectors known in the art, such as various commercially available plasmids, cosmids, phages, retroviruses, and the like, can be used. When the high-temperature-resistant isocitrate dehydrogenase is produced, a gene sequence of the high-temperature-resistant isocitrate dehydrogenase can be connected with an expression regulatory sequence, so that a high-temperature-resistant isocitrate dehydrogenase expression vector is formed. Expression vectors contain an origin of replication and expression control sequences, promoters, enhancers and necessary processing information sites. The expression vector must also contain alternative marker genes, such as a) proteins that provide resistance to antibiotics or other toxic substances (ampicillin, kanamycin, methotrexate, etc.) or b) complementary auxotrophic proteins or c) proteins that provide essential nutrients not present in the complex medium. Suitable marker genes for a variety of different hosts are well known in the art or are set forth in the manufacturer's instructions. Such expression vectors can be prepared by recombinant DNA techniques well known to those skilled in the art, for example, see Sambrook et al, 1989 or Ausubel et al, 1992.
Recombinant expression vectors can be introduced into host cells by methods well known in the art, including: electrical transformation, calcium chloride, particle gun, etc. The process of introducing an exogenous recombinant vector into a host cell is referred to as "transformation". The desired protein is induced to be expressed by culturing the host cell and is obtained by protein isolation techniques well known in the art, such as column chromatography and the like. The protein can also be synthesized artificially by solid phase technique.
In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Prokaryotic cells commonly used are, for example, Escherichia coli, Bacillus subtilis, etc. Commonly used eukaryotic cells such as yeast cells, or various animal and plant cells.
The high-temperature-resistant isocitrate dehydrogenase gene full-length sequence or the fragment thereof can be obtained by a Polymerase Chain Reaction (PCR) amplification method, a recombination method or an artificial synthesis method. For the PCR amplification method, primers can be designed based on the nucleotide sequences disclosed herein, and the entire genomic DNA of Thermoanaerobacterium thermophilum prepared by a conventional method known to those skilled in the art is used as a template to amplify the primers to obtain the sequences. Once the sequence of interest has been obtained, it can be cloned into a vector of interest, transformed into a host cell, and isolated from the propagated host cell by conventional methods to obtain a large batch of the sequence of interest.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.
Example 1: construction of sequencing libraries
The sequencing library is constructed by a whole genome shotgun method (shotgun), firstly culturing thermophilic anaerobes, collecting bacteria by a Marmur (1961) method according to a modified MB culture medium (Balch et al, 1979) of Yanfen Xue (Yanfen Xue, 2000), extracting total DNA, in order to ensure the randomness of the construction of the sequencing library and avoid the problem of generating breaking hot spots to the maximum extent, adopting various methods and different conditions of library construction principles, firstly adopting a physical shearing method (including ultrasonic method and shearing by a Hydroshear Machine), secondly adopting AluI to perform random partial enzyme digestion according to the characteristics of the genome of the bacteria, treating samples with different strengths during physical shearing, treating the samples by setting a sequencing enzyme quantity gradient during enzyme digestion, after the treated samples are subjected to flat end treatment, collecting 1.5-4kb DNA fragments by electrophoresis, connecting the 1.5-4kb DNA fragments with pUCsma 18 which is dephosphorylated, facilitating the connection of the products through an electrotransformation E. coli 5 α, constructing Sagang, and simultaneously connecting the two fragmented DNA fragments which are subjected to overlap connection through a restriction enzyme library (10. the electrophoresis) to obtain a large restriction fragment, and connecting the library which is constructed by a restriction enzyme library (10. about AI).
Example 2: genome sequencing
When the sequencing of the Tengchong thermophilic anaerobic bacteria genome is finished, two full-automatic sequencers are mainly used: ABI377 and MegaBACE 1000. Both sequencers use the principle of electrophoresis for sequencing (see FIG. 2), and 96 samples can be completed at a time. ABI377 is a product of PE company and is one of ABI series. It belongs to a plate gel electrophoresis sequencer. MegaBACE 1000 is a product of FamaXia corporation and belongs to a capillary gel electrophoresis sequencer.
Example 3: basecalling and sequencing quality monitoring
Basecalling refers to the process of obtaining the correct base sequence from the original data file obtained from the sequencer. Because the intensity variation tracks (trace) of light with different wavelengths corresponding to four bases of A, T, G and C are obtained on the sequencer, a computer is required to adopt a certain algorithm to correctly identify the bases corresponding to different tracks. We used Phred software (Ewing B, Hillier L, 1998) because the results were more reliable and the results output more convenient for further analysis by other programs in the same package.
The algorithm principle of performing Basecalling on Phred is that the type of a base is judged according to factors such as the shape, the distance and the signal-to-noise ratio of each peak in a track, and credibility information, namely the sequencing quality of the base, is given to the base. In large-scale sequencing, monitoring of sequencing quality is very important, and directly influences the decision on sequencing, including library construction and coverage rate. Meanwhile, errors possibly occurring in a sequencing experiment can be fed back in time.
Example 4: sequence splicing
Sequence splicing is the assembly of sample sequences obtained by whole genome shotshell method, also called shotgun method random sequencing, into continuous contigs (contigs), and mainly uses the overlapping sequences between them as reference. Considering the influence of the vector present in sequencing, it is necessary to first perform a decoarrier treatment on the sample sequence. The software cross _ match used here and the software Phrap used for the following splices are both the software of Washington university (Gordon D, Abajian C, 1998) in the united states, the basic principle of which is the switch-Waterman algorithm (WatermanMS, 1990). This is a dynamic algorithm that, after considering the comparison between two sequences, can yield a set of consensus sequences of sequences (consensus sequences). The sample sequence after removal of the vector was spliced again with Phrap. The sequencing quality of the bases is also taken into account during splicing, and the confidence level of each base of the obtained public sequence is calculated from the sequencing quality of the sample constituting the public sequence.
Example 5: gene annotation
After obtaining most of the genome sequence (completing the work Frame map), the genome needs to be annotated, including the prediction of Open Reading Frame (ORF), the prediction of gene function, and the analysis of specific RNA fragments.
In the first step, the gene coding sequence was predicted using default parameters of GLIMMER2.0(Delcher, A.L., Harmon, D.1999) and ORPHEUS (Frishman, D.1998) software, and then all predicted open reading frames and non-coding regions (endogenous regions) were compared with the NCBI's non-redundant protein database (non-redundant protein database) using BLAST software (Altschul, S.F.et al.1997) to find genes that might be missed. In determining the starting point of a gene, various information such as sequence homology, ribosome binding site, possible signal peptide sequence and promoter sequence, etc. are referred to. If multiple promoters are present in an open reading frame, the first promoter is generally used as the starting point for the gene. A transcriptional terminator independent of Rho (Rho) factor was predicted in non-coding regions using TransTerm software (Ermolaeva, M.D. 2000). If the terminator is located too far downstream of a gene, it may suggest that the loss of a minigene or sequencing errors artificially shortened the gene, which may serve as a reference for further analysis. In determining frame shift and point mutations, the determination is based primarily on similarity to proteins in the database. If a protein corresponding to two coding sequences adjacent to each other is present, it is considered to be an inactive gene (pseudogenes), since it indicates that the two coding sequences are abnormally suspended due to mutation, thereby inactivating the gene. All analytical results were manually analyzed using the artemissence viewer software (Rutherford, k.et al.2000). Some open reading frames that clearly overlap with other coding sequences, those less than 150 base pairs in length and that have no homology in existing databases and in which there is no obvious promoter or termination region will be removed.
Functional fragments (motif) and functional regions (domains) of the protein were analyzed by comparison with Pfam, PRINTS, PROSITE, ProDom and SMART databases, respectively, and the results were summarized using InterPro database (Apweiler, R.et al 2001). The functional classification of proteins in the COGs classification and the possible metabolic pathways are determined from the NCBI's COGs database (Tatusov, R.L.et al.2001) and with reference to the query results of other databases. The membrane proteins, ABC transporters and transmembrane domains were confirmed using TMHMM software (Krogh, A.et al 2001). Using gram-negative bacteria as parameters, the signal peptide region was analyzed using SIGNALP2.0 software (Nielsen, H.et al 1999).
(4) Hole filling
After the working frame map of the genome is completed, more difficult hole filling work is performed, i.e., 100% of the whole genome is sequenced to obtain a circular genome. The main task is to connect the previously obtained contigs. The method mainly comprises the following steps:
A. using forward and reverse sequencing sample information in sequencing
In the sequencing process, two-way sequencing is performed on some samples intentionally, namely, two ends of an insert are sequenced simultaneously, and the obtained sequence is spliced with other sequences. Because the relation of the pair of sequences on the genome is certain, and the distance between the sequences is approximately known, according to the information, whether a certain contig is reliable or not can be confirmed, and when the pair of sequences are respectively positioned on different contigs, the direction relation and the position relation of the two contigs can be determined, so that reference is provided for further designing experiments.
B. Long insert and Cosmid end sequencing
Based on the same principle, we can construct a library of inserts of different lengths, sequence only the two ends, then splice and analyze the specific positions. These libraries include long insert libraries of 9-12Kb in length and Cosmid libraries around 20-40 Kb. The specific analysis method is as described above.
PCR and end extension Walking experiments
Further biochemical experiments can be performed based on the contig orientations and positional relationships provided by a and B. For example, a pair of primers is designed for PCR amplification, or a primer is synthesized with a certain contig end sequence for end extension (Walking) to fill in holes.
Example 6: preparation and purification of isocitrate dehydrogenase
Designing a primer capable of amplifying a complete coding reading frame according to a full-length isocitrate dehydrogenase coding sequence (SEQ ID NO.1) obtained by gene annotation in the embodiment, and respectively introducing restriction endonuclease sites on positive and negative primers to construct an expression vector, recombining the plasmid DNA of the sequencing library obtained in the embodiment 1 as a template into a pGEX-2T vector (Pharmacia, Piscataway, NJ) under the premise of ensuring correct reading frame after PCR amplification, and then transforming the recombinant vector into Escherichia coli DH5 α (the transformation method is CaCL)2Method or electric transformation method), screening the identified engineering bacteria DH5 α -pGEX-2T-Icd containing expression vectors.
Selecting engineering bacteria DH5 α -pGEX-2T-Icd of single colony, shaking-culturing in 3ml LB culture medium containing 100 ug/ml ampicillin overnight at 37 deg.C, sucking culture solution at 1: 100 concentration, and culturing in new LB culture medium (containing 100 ug/ml ampicillin) for about 3 hr to OD600After reaching 0.5, IPTG was added to a final concentration of 1mmol/L and incubation was continued at 37 ℃ for 0, 1, 2, 3 hours, respectively. Centrifuging 1ml of bacterial liquid with different culture time, adding lysis solution (50 μ l of 2 xSDS loading buffer, 45 μ l of distilled water, 5 μ l of dimercaptoethanol) into bacterial precipitate, suspending bacterial precipitate, decocting in boiling water bath for 5 min, centrifuging at 10000rpmFor 1 min, the supernatant was run on a 12% SDS-PAGE gel. After dyeing, observing that the expected molecular weight and protein amount increase along with the increase of IPTG induction time, namely the strain is the engineering bacteria for expressing the needed protein.
After the engineering bacteria of the needed protein is induced and expressed according to the method, the bacteria are centrifuged and precipitated, 20ml of 50 percent glutathione Sepharose 4B saturated by PBS is added into every 400ml of bacteria, the bacteria are shaken and combined for 30 minutes at 37 ℃, and the bacteria are centrifuged for 10 minutes at 10000rpm to precipitate the glutathione Sepharose 4B combined with the needed protein, and the supernatant is discarded. Adding 100 mul of reduced glutathione eluent into the sediment obtained by every milliliter of ultrasonic liquid, standing for 10 minutes at room temperature, and obtaining the supernatant as the eluted protein. The elution was repeated twice. The eluted supernatant was stored at-80%, subjected to SDS-PAGE, and the purification effect was examined. The protein band at 46061 daltons is another isocitrate dehydrogenase.
Sequence listing
1.SEQ ID NO.1
(1) Sequence characteristics:
a. length: 1212 base pair
b. Type (2): DNA
c. Chain type: double chain
d. The geometrical structure is as follows: linearity
(2) Molecular type: nucleotide, its preparation and use
(3) Description of the sequence:
atggcacaaaaaattgagatgaaggttcctattgtggaaatggacggagatgaaatgaca
agaattatatggaaattgataaaagaaatgcttataaagccctatgtagatttaaagaca
gagtactatgatttgggcattaaaaacagagatgaaacagaagaccaggtcacagttgat
gcagcatacgcaataaaaaaatacggagtcggggtaaaatgcgcaacaatcaccccaaat
gctgaaagagtaaaagaatacaatttgaaaaatatgtggaaaagccctaacgctactata
cgatccatactcgatgggacagtatttcgcacacctattctggttgaaggaataaagccg
cttgtaaggacatggaaaaaacctattacaattgcaaggcatgcttatggggatatttat
aaaggggtagaatacagaatccctgaaaaaggtaaggctgagcttgtctttacttctgaa
aaaggagaagagataaggtatgcaatacacgagtttgaaactcctggtgtcatattaggt
atgcacaacactgatgagtcaataaaaagttttgcgagagcatgttttaattatgcttta
gatacgaagcaggatttgtggtttgcgactaaagacaccatttcaaaaatatacgaccac
agatttaaagacatattccaggaaatatatgaaaatgaatataaagaaaaatttgaagaa
gcaggtattgaatatttttacacccttattgacgatgccgtagctcgaattgtaaggtct
gaaggcggaatgatttgggcatgcaaaaattatgacggcgatgtaatgtcagatatggta
gcaacagcatttggcagccttgccatgatgacttctgtactggtatctcctgatggtaaa
tacgaatttgaagcggctcatgggacagttacaaggcattattataaataccttaaaggt
gaagaaacttctactaatcctattgctacaattttcgcgtggacaggtgctttgaaaaag
agaggagaacttgacggaataaaagaccttgttgattttgcagataagcttgaaaaagcc
tcacttaaaacggttgaaaaaggaataatgacaaaagacttagccgctttatctgaattg
ccaaataaaactgttgtaaacacggaaactttcctcattgaaattaaaaaaactttagaa
gagatgctgtaa
2.SEQ ID NO.2
(1) sequence characteristics:
a. length: 403 amino acid
b. Type (2): polypeptides
c. Chain type: single strand
d. The geometrical structure is as follows: stereo
(2) Molecular type: protein
(3) Description of sequences
MAQKIEMKVPIVEMDGDEMTRIIWKLIKEMLIKPYVDLKTEYYDLGIKNRDETEDQVTVD
AAYAIKKYGVGVKCATITPNAERVKEYNLKNMWKSPNATIRSILDGTVFRTPILVEGIKP
LVRTWKKPITIARHAYGDIYKGVEYRIPEKGKAELVFTSEKGEEIRYAIHEFETPGVILG
MHNTDESIKSFARACFNYALDTKQDLWFATKDTISKIYDHRFKDIFQEIYENEYKEKFEE
AGIEYFYTLIDDAVARIVRSEGGMIWACKNYDGDVMSDMVATAFGSLAMMTSVLVSPDGK
YEFEAAHGTVTRHYYKYLKGEETSTNPIATIFAWTGALKKRGELDGIKDLVDFADKLEKA
SLKTVEKGIMTKDLAALSELPNKTVVNTETFLIEIKKTLEEML
Claims (7)
1. An isolated DNA molecule characterized by: the nucleotide sequence of the polypeptide with the activity of high-temperature resistant isocitrate dehydrogenase protein is coded as the same as the nucleotide sequence of SEQ ID NO. 1.
2. The DNA molecule of claim 1, wherein: said nucleotide sequence encodes a polypeptide having the amino acid sequence in SEQ. ID No.2 or a modified form of said polypeptide which is functionally equivalent or related to a thermostable isocitrate dehydrogenase.
3. An isolated polypeptide, comprising: has the same amino acid sequence as SEQ ID No.2 and has high temperature resistant isocitrate dehydrogenase activity.
4. The polypeptide of claim 3, wherein: it has the polypeptide of the amino acid sequence in SEQ ID No. 2.
5. A carrier, characterized by: it contains the DNA of claim 1.
6. A host cell, characterized in that: it is a prokaryotic or eukaryotic cell transformed with the vector of claim 5.
7. The method for preparing the high-temperature-resistant isocitrate dehydrogenase is characterized by comprising the following steps of:
1) separating out a nucleotide sequence SEQ ID NO.1 of a gene coding a high-temperature-resistant isocitrate dehydrogenase;
2) constructing an expression vector containing a nucleotide sequence of SEQ ID NO. 1;
3) transferring the expression vector in the step 2) into a host cell to form a recombinant cell capable of producing high-temperature-resistant isocitrate dehydrogenase;
4) culturing the recombinant cells in step 3);
5) separating and purifying to obtain the high-temperature-resistant isocitrate dehydrogenase.
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| CN1166779C true CN1166779C (en) | 2004-09-15 |
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| CN101157955B (en) * | 2007-10-09 | 2011-03-16 | 华中农业大学 | Pig isocitric acid dehydrogenase gene IDH3-gamma as genetic marker of production trait in pigs |
| CN116426512B (en) * | 2022-08-11 | 2023-09-26 | 海南师范大学 | Salt-tolerant (NaCl) isocitrate enzyme gene and application thereof |
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