CN1379095A - High-temp. resistant exo-polyphosphate enzyme gene and its coded polypeptide and preparing process - Google Patents
High-temp. resistant exo-polyphosphate enzyme gene and its coded polypeptide and preparing process Download PDFInfo
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- CN1379095A CN1379095A CN 02111357 CN02111357A CN1379095A CN 1379095 A CN1379095 A CN 1379095A CN 02111357 CN02111357 CN 02111357 CN 02111357 A CN02111357 A CN 02111357A CN 1379095 A CN1379095 A CN 1379095A
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Abstract
The invention discloses a high-temperature-resistant exo-polyphosphate enzyme gene, a polypeptide coded by the same and a preparation method thereof. It relates to a method for coding separated DNA with high-temp. resisting exopolyphosphate enzyme activity or its functional equivalent variant and utilizing recombinant DNA technology to produce polypeptide with high-temp. resisting exopolyphosphate enzyme activity or its functional equivalent variant by using the described separated DNA. Based on sequencing and analysis of Tengchong thermophilic anaerobe whole genome, high temperature resistant exo-polyphosphate enzyme gene is cloned and separated. The gene is used to produce high temperature resistant exo-polyphosphatase transgenic microbe or transgenic animal and plant, and the enzyme coded by the gene is recovered. In addition, the invention also provides an amino acid sequence and a functional equivalent of the polypeptide with the high-temperature-resistant exopolyphosphate enzyme activity. Also provided are methods for preparing, isolating, and purifying polypeptides having high temperature-resistant exopolyphosphatase activity.
Description
Technical field
The present invention relates to sudden change or genetic engineering, relate in particular to a kind of high temperature resistant circumscribed Tripyrophosphoric acid enzyme gene and encoded polypeptides and preparation method.
Background technology
The effect of circumscribed Tripyrophosphoric acid enzyme (exopolyphosphatases) is catalysis such as afterreaction N{ poly-phosphate }+H (
2) O=(N-1) { poly-phosphate }+phosphoric acid salt.
Research has now disclosed the polyphosphate (understanding) of inorganics from bacterium and yeast, belong to the orthophosphoric acid salt residue, is a kind of polymer of high-energy linearity, and simultaneously, it also is present in the high eukaryotic cell., it is the basis of metabolic enzyme in the mammiferous cell, remains not understanding by the people.Evidence suggests that what extract for the first time is that alkali Phosphoric acid esterase from little Roll is a kind of circumscribed Tripyrophosphoric acid enzyme.
The circumscribed Tripyrophosphoric acid endonuclease capable branch phosphorus molecule that depolymerizes, in the suitableeest pH value was alkaline range, the positively charged ion of divalent was catalytic dispensable on.But it can suppress the degraded of polymerization phosphorus.No matter be that along with the length that increases polymer chain, the special activity of this enzyme can reduce in the PH scope of alkalescence and nature.
Inorganic polyphosphate is the polymkeric substance of a linearity, has all found the existence of this enzyme in each tested organism.Polymerization phosphorus has indicative function (because this process has the generation of polymerization phosphorus) in the regulation and control of genetic expression, reduce to almost can not found concentration for the content of the polymerization phosphorus of cell in intestinal bacteria, and its reason is because the synthetic of the circumscribed Tripyrophosphoric acid enzyme of over-drastic arranged.Simultaneously, discover that the excessive synthetic cell of circumscribed Tripyrophosphoric acid enzyme splits the plain C sensitivity of enzyme to UV or silk more.Split the plain C processing of enzyme with silk after, find the accumulation of polymerization phosphorus.
Polyphosphate has been found the effect of the formation that suppresses ATP.Its effect is to bring into play by the mode of the competition of adenylic acid (AMP) nucleosides.And the effect of the polyphosphate of long-chain is stronger than short phosphatic restraining effect.This has just disclosed, and this circumscribed Tripyrophosphoric acid enzyme has the metabolic effect of the polyphosphate an of the unknown promptly to regulate the spissated effect of intracellular adenylic acid (AMP) nucleosides.
In sum, circumscribed Tripyrophosphoric acid enzyme has the depolymerize function of phosphorus of branch, thereby this enzyme can be used for metabolism class drug research, the research and development of the medicine that generates at ATP and as the indication research of gene expression regulation with use.
The Tengchong thermophilc anaerobe that the present invention relates to (Thermoanaerobacter tangcongensis), it is a kind of microorganism that lives in the hot spring of Yunnan Province of China province Tengchong County, it is a kind of thermophilic eubacterium (eubacteria), optimum growth temperature is 75 degrees centigrade, anaerobic growth, the gramstaining reaction is positive.It is at first to be found and carried out the analysis on the taxonomy by Microbe Inst., Chinese Academy of Sciences.Bacterial classification is kept at Chinese microorganism and preserves center MB4
T(Chinese collection of microorganisms AS 1.2430
T=JCM 11007
T).This thermophilc anaerobe is the distinctive species of China, and the high temperature resistant circumscribed Tripyrophosphoric acid enzyme that is had in its body also has own its specific structure.
Summary of the invention
One of purpose of the present invention provides a kind of isolating, and coding has the nucleotide sequence of the polypeptide of high temperature resistant circumscribed Tripyrophosphoric acid enzymic activity.
Two of purpose of the present invention provides a kind of isolating, has high temperature resistant circumscribed Tripyrophosphoric acid peptide activity.
Purpose of the present invention also provide the DNA that contains the high temperature resistant circumscribed Tripyrophosphoric acid enzyme of encoding recombinant vectors, contain the host cell of aforementioned recombinant vectors, and this proteic method of production.
A first aspect of the present invention provides a kind of coding to have the nucleotide sequence of the polypeptide of high temperature resistant circumscribed Tripyrophosphoric acid enzymic activity.This nucleotide sequence can encode the polypeptide with the aminoacid sequence among the sequence table SEQ ID NO.2 or the modified forms of described polypeptide, on this modified forms function quite or relevant with circumscribed Tripyrophosphoric acid enzyme.Nucleotide sequence has the polynucleotide sequence of sequence table SEQ ID NO.1 and its mutant form, and mutation type comprises: disappearance, nonsense, insertion, missense.
A second aspect of the present invention provides a kind of polypeptide of high temperature resistant circumscribed Tripyrophosphoric acid enzymic activity.This polypeptide has polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of the aminoacid sequence among the sequence table SEQ ID No.2.
The present invention also provides the method for producing high temperature resistant circumscribed Tripyrophosphoric acid enzyme:
1) isolates the nucleotide sequence SEQ ID NO.1 of gene of the high temperature resistant circumscribed Tripyrophosphoric acid enzyme of coding;
2) make up the expression vector that contains SEQ ID NO.1 nucleotide sequence;
3) with step 2) in expression vector change host cell over to, form the reconstitution cell can produce high temperature resistant circumscribed Tripyrophosphoric acid enzyme;
4) culturing step 3) in reconstitution cell;
5) separation, purifying obtain high temperature resistant circumscribed Tripyrophosphoric acid enzyme.
The present invention relates to the separation and the expression of the circumscribed Tripyrophosphoric acid enzyme gene of thermophilc anaerobe.Based on Tengchong thermophilc anaerobe genome sequencing and analysis, clone and separate high temperature resistant circumscribed Tripyrophosphoric acid enzyme gene.Use the transgenic microorganism or the animals and plants of the high temperature resistant circumscribed Tripyrophosphoric acid enzyme of this genes produce, and reclaim the enzyme that obtains this genes encoding.In addition, the present invention also provides amino acid sequence of polypeptide and the functional equivalent body with high temperature resistant circumscribed Tripyrophosphoric acid enzymic activity.Simultaneously, the present invention also provides preparation, separates, and purifying has the method for the polypeptide of high temperature resistant circumscribed Tripyrophosphoric acid enzymic activity.
Description of drawings
Fig. 1 is an order-checking library construction flow chart of steps;
Fig. 2 is order-checking and data analysis schema.
Embodiment
At first, the invention provides isolating, the nucleic acid molecule of the polypeptide of the high temperature resistant circumscribed Tripyrophosphoric acid enzymic activity of encoding, this nucleic acid molecule has the nucleotide sequence shown in the sequence table SEQ .ID NO.1 by Tengchong thermophilc anaerobe genome sequencing and analysis are obtained.It has been encoded and has had 301 the amino acid whose polypeptide that have of high temperature resistant circumscribed Tripyrophosphoric acid enzymic activity, and the supposition molecular weight of this polypeptide is 33477 dalton.The invention still further relates to a kind of recombinant vectors, this carrier comprises isolating nucleic acid molecule of the present invention, and the host cell that includes recombinant vectors.Simultaneously, the present invention includes the method that makes up this recombinant vectors and host cell, and utilize the recombined engineering technology to produce the method for circumscribed Tripyrophosphoric acid enzyme.
The present invention provides a kind of isolating high temperature resistant circumscribed Tripyrophosphoric acid enzyme or polypeptide further, it is characterized in that having the aminoacid sequence shown in the sequence table SEQ .ID NO.2, or at least 70% is similar, more preferably, have at least 90%, 95%, 99% identical.
In the present invention, " isolating " DNA is meant that this DNA or fragment have been arranged in its both sides under native state sequence separates, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.
In the present invention, " high temperature resistant circumscribed Tripyrophosphoric acid enzyme gene " refer to the encode nucleotide sequence of polypeptide with high temperature resistant circumscribed Tripyrophosphoric acid enzymic activity is as nucleotide sequence and the degenerate sequence thereof of sequence table SEQ .ID NO.1.This degenerate sequence be meant have one or more codons to be encoded in this sequence the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of known codon, so be low to moderate about 70% the degenerate sequence described aminoacid sequence of sequence table SEQ ID NO.2 of also encoding out with SEQ IDNO.1 nucleotide sequence homology.This term also comprises can be under the rigorous condition of moderate, more preferably under highly rigorous condition with the nucleotide sequence of the nucleotide sequence hybridization of sequence table SEQ ID NO.1.This term also comprises and sequence table SEQ ID NO.1 nucleotide sequence homology 70% at least, preferably at least 80%, more preferably at least 90%, and at least 95% nucleotide sequence best.
In the present invention, " isolating " proteic polypeptide is meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as uses column chromatography, and PAGE or HPLC method are measured the purity of polypeptide.Isolated polypeptide is substantially free of the component of following it under the native state.
In the present invention, " high temperature resistant circumscribed Tripyrophosphoric acid enzyme " refers to have the sequence table SEQ ID NO.2 polypeptide of sequence of high temperature resistant circumscribed Tripyrophosphoric acid enzymic activity.This term also comprises the varient of sequence table SEQ ID NO.2 sequence, and these varients have and natural high temperature resistant circumscribed Tripyrophosphoric acid enzyme identical functions.These varients include, but is not limited to several amino acid whose disappearances, insert and/or replace, and add one or several amino acid at C latter end and/or N-terminal, also can be the difference that does not influence on the modified forms of sequence.For example, for known in the field, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C latter end and/or N-terminal and also can not change proteinic function usually.This term also comprises the active fragments and the reactive derivative of high temperature resistant circumscribed Tripyrophosphoric acid enzyme.
In the present invention, can select various carrier known in the art for use, as various plasmids, clay, phage and the retrovirus of selling on the market etc.When producing high temperature resistant circumscribed Tripyrophosphoric acid enzyme of the present invention, high temperature resistant circumscribed Tripyrophosphoric acid enzyme gene order operationally can be connected in expression regulation sequence, thereby form high temperature resistant circumscribed Tripyrophosphoric acid expression of enzymes carrier.This expression vector contains replication origin and expression regulation sequence, promotor, enhanser and necessary machining information site.Expression vector also must contain alternative marker gene, as a) providing to microbiotic or other toxicant (penbritin, the protein or the b of resistance kantlex, methotrexate etc.)) complementary auxotroph protein or c) protein of the essential nutritive ingredient that does not have in the complex medium is provided.Various different hosts' appropriate flags gene is well known in the art or production firm's specification sheets indicates.These expression vectors can be with recombinant DNA technology known in those skilled in the art preparation, as can be with reference to people's such as Sambrook way (1989), or people's such as Ausubel way (1992).Recombinant expression vector can be introduced host cell with method well known in the art, and these methods comprise: electrotransformation, Calcium Chloride Method, particle bombardment etc.The process that the external source recombinant vectors is imported host cell is called " conversion ".By cultivating host cell, induce the expression of desirable proteins, and by protein separation technology known in the art, obtain required protein as column chromatography etc.Also can adopt these protein of synthetic such as solid phase technique.In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.Prokaryotic cell prokaryocyte such as intestinal bacteria commonly used, Bacillus subtilus etc.Eukaryotic cell such as yeast cell commonly used, or various animal and plant cells.
High temperature resistant circumscribed Tripyrophosphoric acid enzyme full length gene sequence of the present invention or its fragment can be used PCR (polymerase chain reaction) TRAP usually, recombination method, or the method for synthetic obtains.For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence design primer, is template with the thermophilc anaerobe complete genome DNA of ordinary method preparation well known by persons skilled in the art, increases and obtains relevant sequence.In case obtained relevant sequence, just it can be cloned into relevant carrier, change host cell again over to, from the host cell after the propagation, separate obtaining large batch of relevant sequence then by ordinary method.
Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: make up the order-checking library
The structure in order-checking library adopts full genome shotgun approach (shotgun) to carry out.At first cultivate the Tengchong thermophilc anaerobe, cultural method is pressed Marmur (1961) method and is collected bacterium by (Yanfen Xue, 2000) improved MB substratum (Balch et al., 1979), extracts total DNA.For the randomness of the library construction that guarantees to check order, farthest avoid producing the problem of breakage hot spot, that adopts several different methods, different condition builds the storehouse principle.Adopt earlier physics cutting method (comprise supersonic method and shear with Hydroshear Machine), next is selected for use AluI to carry out the random partial enzyme according to this bacterium genome signature and cuts.Adopt varying strength to handle sample when physics is sheared, handle sample by enzyme amount gradient is set when enzyme is cut.Sample after the processing adopts electrophoresis fraction collection 1.5-4kb dna fragmentation after flat terminal the processing, be connected with the dephosphorylized pUC18 that cuts through the SmaI enzyme, connects product has made up random sequencing by electric Transformed E .coli DH5 α library.Simultaneously, (cut genomic dna in the order-checking library that has also made up long insertion fragment (about 10kb) for the ease of the later overlap joint of contig (contig) with Sau3AI random partial enzyme, electrophoresis is collected the fragment about 10kb, is connected, makes up the library with the dephosphorylized pUC18 that cuts through the BamHI enzyme).The order-checking of these two ends in library can obtain the relation between the contig (contig) in structure is finished the process of figure (finishing), and can solve the difficulty that cause filling-up hole in bigger hole (gap).Build the storehouse flow process and see Fig. 1.
Embodiment 2: gene order-checking
When finishing the genomic order-checking of Tengchong thermophilc anaerobe, two kinds of full-automatic sequenator: ABI377 and MegaBACE 1000 have mainly been used.These two kinds of sequenators all are to utilize the principle of electrophoresis (see figure 2) that checks order, and can finish 96 samples at every turn.ABI377 is the product of ABI company, is a kind of of ABI series.It belongs to the plate gel electrophoresis sequenator.MegaBACE 1000 is products of Pharmacia Corp, belongs to the capillary gel electrophoresis sequenator.
Embodiment 3: data analysis
1) Basecalling and sequencing quality monitoring
So-called Basecalling is meant the process that obtains correct base sequence from the raw data file that sequenator obtains.Because that obtain on the sequenator is A, T, G, the light intensity variation track (trace) of the different wave length that four kinds of base pairs of C are answered need take certain algorithm therefrom correctly to identify the base of different track correspondences with computer.What we used is Phred software (Ewing B, Hillier L, 1998), and reason is that its result is more reliable, and other programs that its result exports in the same software package of being more convenient for are further analyzed.
Phred carries out the algorithm principle of Basecalling, is the shape according to each peak in the track, and spacing, and factor such as signal to noise ratio are judged the base type, simultaneously this base are provided reliability information, i.e. the sequencing quality of base.In large scale sequencing, the monitoring of sequencing quality is crucial, and it directly influences the decision-making to order-checking, comprises the structure in library, the size of fraction of coverage.Can in time feed back the error that may occur in the order-checking experiment simultaneously.
2) sequence assembly
So-called sequence assembly, the sample sequence that full genome shotgun approach (claiming shotgun again) random sequencing is obtained is assembled into successive contig (contig) exactly, mainly utilizes the overlap between them for referencial use.Consider the influence that has carrier in the order-checking, need earlier sample sequence to be unloaded body and handle.Here used software cross_match and the back used software Phrap of splicing are the software (Gordon D, Abajian C, 1998) of U.S. Washington university, and its ultimate principle is Swith-Waterman algorithm (WatermanMS, 1990).This is a kind of dynamic algorithm, after having considered the comparison between the sequence in twos, can obtain the publicly-owned sequence (consensus sequence) of one group of sequence.Sample sequence behind the removal carrier splices with Phrap again.In when splicing, the sequencing quality of base also has been considered, and the confidence level of resulting publicly-owned each base of sequence is calculated by the sequencing quality of the sample of forming this publicly-owned sequence.
3) gene annotation
After obtaining genomic most of sequence (frame diagram of finishing the work) substantially, just need carry out note, comprise and carry out open reading frame (Open Reading Frame, prediction ORF) genome, the prediction of gene function, and the segmental analysis of special RNA etc.
The first step adopts the GLIMMER2.0 (Delcher of default parameter, A.L., Harmon, D.1999) and ORPHEUS (Frishman, D.1998) software prediction gene coded sequence, open frame and the non-coding region (intergenic region) of all predictions all use BLAST software (Altschul, S.F.et al.1997) and the irredundant albumen database (non-redundant proteindatabase) of NCBI (American National biotechnology information center) relatively to find the gene that may miss then.When judging the starting point of a gene, will be with reference to various relevant informations, as sequence homology, ribosome bind site, possible signal peptide sequence and promoter sequence etc.If when in an open reading frame, a plurality of promotor occurring, generally adopt the starting point of first promotor as gene.(Ermolaeva M.D.2000) predicts the transcription terminator that does not rely on Rho (ρ) factor at non-coding region to adopt TransTerm software.If this terminator be positioned at a gene the catchment too at a distance, then may hint a minigene lose or the mistake that checks order has shortened this gene artificially, can be used as the reference of further analysis.When determining to move frame sudden change and point mutation, main basis is judged with the proteinic similarity in the database.If protein is corresponding to the situation of two encoding sequences adjacent one another are, then be considered to a non-activity gene (pseudogene seudogenes), produce the abort phenomenon because this illustrates between these two encoding sequences owing to suddenling change, and then gene is lost activity.All analytical resultss use Artemis sequence viewer software (Rutherford, K.et al.2000) to carry out manual analysis again.Some are obviously shown eclipsed with other code sequence and open frame, and length does not have homology and wherein do not have tangible promotor or open reading frame that termination is regional will be removed less than 150 base pairs and in the data with existing storehouse.
Proteinic function fragment (motif) and functional area (domain) employing and Pfam, PRINTS, PROSITE, ProDom and SMART database respectively compare, the result uses InterPro database (Apweiler, R.et al.2001) to carry out Macro or mass analysis again.According to the COGs database (Tatusov, R.L.et al.2001) of NCBI and with reference to other result of querying database determine protein in the COGs classification functional classification and possible pathways metabolism.Confirm membranin, abc transport albumen and stride the film functional domain with TMHMM software (Krogh, A.et al.2001).The employing Gram-negative bacteria is a parameter, with SIGNALP2.0 software (Nielsen, H.et al.1999) analytical signal peptide zone.
4) filling-up hole
After finishing genomic work frame chart, will carry out the filling-up hole work of difficulty more, promptly finish the order-checking of whole genome 100%, obtain an annular genome.Groundwork is exactly that the contig that obtains is previously coupled together.This is a very concrete and numerous and diverse job.Main method comprises:
A. utilize the forward and reverse order-checking sample message in the order-checking
In the order-checking process, we have carried out two-way order-checking to some sample intentionally, check order simultaneously promptly that certain inserts segmental two ends, institute's calling sequence are spliced with other sequences again.Because the relation of this a pair of sequence on genome is certain, distance between them is roughly known, according to this information, one can confirm whether certain section contig is reliable, the 2nd, when this a pair of sequence lays respectively on the different contig, can determine direction relations and the position relation of these two contig, for further contrived experiment provides reference.
B. long fragment and equipment type carrier clay (Cosmid) end sequencing of inserting
Based on same principle, we can make up the insertion fragment library of different lengths, and only to its two ends order-checking, its particular location is analyzed in splicing then.These libraries comprise that length is the long Cosmid library of inserting about sheet phase library and 20-40Kb of 9-12Kb.Specific analytical method is same as above.
C.PCR and terminal extend (Walking) test
The direction of the contig that is provided according to above-mentioned steps A and B and position relation, further Biochemistry Experiment just can have been carried out.As design a pair of primer and carry out pcr amplification, or carry out end extension (Walking) with a certain contig end sequence synthetic primer and come filling-up hole etc.
Embodiment 4: the preparation of circumscribed Tripyrophosphoric acid enzyme and purification
The circumscribed Tripyrophosphoric acid enzyme full length gene encoding sequence (SEQ IDNO.1) that obtains according to gene annotation among the embodiment, design and to amplify the primer that complete coding is read frame, and on positive anti-primer, introduce restriction endonuclease sites respectively, so that construction of expression vector.Plasmid DNA with the order-checking library that obtains among the embodiment 1 is a template, behind pcr amplification, guarantee to read recombinate under the correct prerequisite of frame to the pGEX-2T carrier (Pharmacia, Piscataway, NJ).Again recombinant vectors is transformed into that (method for transformation is CaCL in the bacillus coli DH 5 alpha
2Method or electrotransformation).Screening and Identification to the engineering bacteria DH5 α-pGEX-2T-GppA that contains expression vector.
The engineering bacteria DH5 α-pGEX-2T-GppA of picking list bacterium colony contains in the LB substratum of 100 μ g/ml penbritins jolting in 3ml and cultivates 37 ℃ and spend the night, draw nutrient solution by 1: 100 concentration and in new LB substratum (containing 100 μ g/ml penbritins), cultivated about 3 hours, to OD
600After reaching 0.5, add IPTG, continue at 37 ℃ and cultivated respectively 0,1,2,3 hours to final concentration 1mmol/L.It is centrifugal to get the different 1ml bacterium liquid of incubation time, in the bacterial precipitation thing, add lysate (2 * SDS sample-loading buffer, 50 μ l, distilled water 45 μ l, 3-mercaptoethanol 5 μ l), the suspendible bacterial precipitation, boiled in the boiling water bath 5 minutes, centrifugal 1 minute of 10000rpm, supernatant adds electrophoresis in the 12%SDS-PAGE glue.The bacterial strain that the protein content of dyeing back observation expection molecular weight size increases with the IPTG induction time is the engineering bacteria of expressing desirable proteins.
As stated above behind the engineering bacteria of abduction delivering desirable proteins, with bacterium centrifugation, add 50% saturated Triptide Sepharose 4B of 20ml PBS by every 400ml bacterium, 37 ℃ of joltings were in conjunction with 30 minutes, 10000rpm precipitated the Triptide Sepharose 4B that combines desirable proteins in centrifugal 10 minutes, abandoned supernatant.Add 100 μ l reduced glutathione elutriants by every milliliter of ultrasonic liquid gained precipitation, room temperature was put 10 minutes, and supernatant is the albumen of wash-out.Repeat twice of wash-out.The supernatant of wash-out is stored in-80 ℃, and carries out SDS-PAGE SEQ ID NO.1 electrophoresis, detects purification effect.Protein band at 33477 dalton place is circumscribed Tripyrophosphoric acid enzyme.
＜110〉＜120〉＜110〉＜120〉＜140〉＜141〉＜160〉2＜170〉PatentIn Version 3.1＜210〉1＜211〉906＜212〉DNA＜213〉 ( Thermoanaerobacter tangcongensis )＜220〉＜221〉gene＜222〉 ( 1 ) ... ( 906 )＜223〉n＝agct＜400〉1atgagagtct ctgcgataga tattggaaca aattctgtca gacaattgat atgcgacgta 60gaagatggca gtttaattag attgcgtaaa gatgtaaaaa taacccgttt aggggaagga 120ttaataaaaa cgggtagact aaaccctggt gctgttcaga gaactgtgga gacgatagag 180gaatttatat caatggcaga gaaggaaggt gtagatgaga tttatgcttt tgctacttct 240gctatgagag aagctaaaaa taaggagcta tttttggata atttttcaaa atttaatata 300gcattagata tactagacgg tgatactgaa agcctatttg gctatatagg tgctgtcaaa 360gggataaaat tggataaagc gttggttatt gatataggag gagggagtac agagttagcc 420tataaaggtt ttgaatttat aaaagaaagt tttggtatag gagcagtgaa acttacagaa 480gaattcgtaa aaagtgaccc tccctcttta gaagaataca acgatatgag gctttacata 540atagatacag tggtaaattc aataggcaag tataatgaaa ttgaaaggct tgttggaatt 600ggagggacta tcacaagttt agcagcagta tcacagcaat tggaaattta ttcggaggaa 660aaagtacacg gatatgttct agagagggag gaaataaaga gaattttaga tagatttatg 720tccgtttctc ttgaggaaag gaaaaaaata gctggtcttc agccagaaag agccgatata 780ataattgctg gaacagctat tttacttact cttttagaaa tatttaataa aaaagaaatt 840actgtaagcg aatgggacaa cctagaagga gcagtgttgt ataagtttgg cggtatgaat 900atataa 906＜210〉2＜211〉301＜212〉PRT＜213〉 ( Thermoanaerobacter tangcongensis )＜400〉2Met Arg Val Ser Ala Ile Asp Ile Gly Thr Asn Ser Val Arg Gln Leu1 5 10 15Ile Cys Asp Val Glu Asp Gly Ser Leu Ile Arg Leu Arg Lys Asp Val
20 25 30Lys?Ile?Thr?Arg?Leu?Gly?Glu?Gly?Leu?Ile?Lys?Thr?Gly?Arg?Leu?Asn
35 40 45Pro?Gly?Ala?Val?Gln?Arg?Thr?Val?Glu?Thr?Ile?Glu?Glu?Phe?Ile?Ser
50 55 60Met?Ala?Glu?Lys?Glu?Gly?Val?Asp?Glu?Ile?Tyr?Ala?Phe?Ala?Thr?Ser65 70 75 80Ala?Met?Arg?Glu?Ala?Lys?Asn?Lys?Glu?Leu?Phe?Leu?Asp?Asn?Phe?Ser
85 90 95Lys?Phe?Asn?Ile?Ala?Leu?Asp?Ile?Leu?Asp?Gly?Asp?Thr?Glu?Ser?Leu
100 105 110Phe?Gly?Tyr?Ile?Gly?Ala?Val?Lys?Gly?Ile?Lys?Leu?Asp?Lys?Ala?Leu
115 120 125Val?Ile?Asp?Ile?Gly?Gly?Gly?Ser?Thr?Glu?Leu?Ala?Tyr?Lys?Gly?Phe
130 135 140Glu?Phe?Ile?Lys?Glu?Ser?Phe?Gly?Ile?Gly?Ala?Val?Lys?Leu?Thr?Glu145 150 155 160Glu?Phe?Val?Lys?Ser?Asp?Pro?Pro?Ser?Leu?Glu?Glu?Tyr?Asn?Asp?Met
165 170 175Arg?Leu?Tyr?Ile?Ile?Asp?Thr?Val?Val?Asn?Ser?Ile?Gly?Lys?Tyr?Asn
180 185 190Glu?Ile?Glu?Arg?Leu?Val?Gly?Ile?Gly?Gly?Thr?Ile?Thr?Ser?Leu?Ala
195 200 205Ala?Val?Ser?Gln?Gln?Leu?Glu?Ile?Tyr?Ser?Glu?Glu?Lys?Val?His?Gly
210 215 220Tyr?Val?Leu?Glu?Arg?Glu?Glu?Ile?Lys?Arg?Ile?Leu?Asp?Arg?Phe?Met225 230 235 240Ser?Val?Ser?Leu?Glu?Glu?Arg?Lys?Lys?Ile?Ala?Gly?Leu?Gln?Pro?Glu
245 250 255Arg?Ala?Asp?Ile?Ile?Ile?Ala?Gly?Thr?Ala?Ile?Leu?Leu?Thr?Leu?Leu
260 265 270Glu?Ile?Phe?Asn?Lys?Lys?Glu?Ile?Thr?Val?Ser?Glu?Trp?Asp?Asn?Leu
275 280 285Glu?Gly?Ala?Val?Leu?Tyr?Lys?Phe?Gly?Gly?Met?Asn?Ile
290 295 300
Claims (8)
1. an isolated DNA molecule is characterized in that: be the nucleotide sequence that coding has the active polypeptide of high temperature resistant circumscribed Tripyrophosphoric acid zymoprotein.
2. dna molecular as claimed in claim 1, it is characterized in that: the polypeptide of the aminoacid sequence among the said nucleotide sequence coded SEQ.ID of the having NO.2 or the modified forms of this polypeptide, on this modified forms function quite or relevant with high temperature resistant circumscribed Tripyrophosphoric acid enzyme.
3. dna molecular as claimed in claim 1 is characterized in that: said nucleotide sequence has the polynucleotide sequence of SEQ ID NO.1 and its mutant form, and mutation type comprises: disappearance, nonsense, insertion, missense.
4. polypeptide separated, it is characterized in that: it has high temperature resistant circumscribed Tripyrophosphoric acid enzymic activity.
5. polypeptide as claimed in claim 4 is characterized in that: it has polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of the aminoacid sequence among the SEQ ID No.2.
6. carrier, it is characterized in that: it contains the DNA in the claim 1.
7. host cell is characterized in that: it is prokaryotic cell prokaryocyte or the eukaryotic cell that transforms with the described carrier of claim 6.
8. a method for preparing high temperature resistant circumscribed Tripyrophosphoric acid enzyme is characterized in that this method comprises the steps:
1) isolates the nucleotide sequence SEQ ID NO.1 of gene of the high temperature resistant circumscribed Tripyrophosphoric acid enzyme of coding;
2) make up the expression vector that contains SEQ ID NO.1 nucleotide sequence;
3) with step 2) in expression vector change host cell over to, form the reconstitution cell can produce high temperature resistant circumscribed Tripyrophosphoric acid enzyme;
4) culturing step 3) in reconstitution cell;
5) separation, purifying obtain high temperature resistant circumscribed Tripyrophosphoric acid enzyme.
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