CN1379093A - High-temperature resistant peptidase E gene and its coded polypeptide and preparing method - Google Patents
High-temperature resistant peptidase E gene and its coded polypeptide and preparing method Download PDFInfo
- Publication number
- CN1379093A CN1379093A CN 02110735 CN02110735A CN1379093A CN 1379093 A CN1379093 A CN 1379093A CN 02110735 CN02110735 CN 02110735 CN 02110735 A CN02110735 A CN 02110735A CN 1379093 A CN1379093 A CN 1379093A
- Authority
- CN
- China
- Prior art keywords
- peptidase
- polypeptide
- refractory
- sequence
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010071005 peptidase E Proteins 0.000 title claims abstract description 43
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 34
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 34
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 34
- 108020004414 DNA Proteins 0.000 claims abstract description 14
- 230000000694 effects Effects 0.000 claims abstract description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 3
- 239000002773 nucleotide Substances 0.000 claims description 21
- 125000003729 nucleotide group Chemical group 0.000 claims description 21
- 210000004027 cell Anatomy 0.000 claims description 18
- 230000008859 change Effects 0.000 claims description 11
- 239000013604 expression vector Substances 0.000 claims description 11
- 239000012634 fragment Substances 0.000 claims description 11
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 238000003780 insertion Methods 0.000 claims description 4
- 230000037431 insertion Effects 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 230000008034 disappearance Effects 0.000 claims description 3
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 3
- 230000035772 mutation Effects 0.000 claims description 3
- 108091033319 polynucleotide Proteins 0.000 claims description 3
- 102000040430 polynucleotide Human genes 0.000 claims description 3
- 239000002157 polynucleotide Substances 0.000 claims description 3
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 102000053602 DNA Human genes 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 36
- 102000004190 Enzymes Human genes 0.000 abstract description 11
- 108090000790 Enzymes Proteins 0.000 abstract description 11
- 238000012163 sequencing technique Methods 0.000 abstract description 10
- 238000004458 analytical method Methods 0.000 abstract description 9
- 238000002360 preparation method Methods 0.000 abstract description 8
- 244000005700 microbiome Species 0.000 abstract description 5
- 241000196324 Embryophyta Species 0.000 abstract description 3
- 241001465754 Metazoa Species 0.000 abstract description 3
- 108020004511 Recombinant DNA Proteins 0.000 abstract description 2
- 230000009261 transgenic effect Effects 0.000 abstract description 2
- 108700026220 vif Genes Proteins 0.000 abstract 1
- 102000004169 proteins and genes Human genes 0.000 description 17
- 150000001413 amino acids Chemical group 0.000 description 16
- 235000018102 proteins Nutrition 0.000 description 15
- 241000894006 Bacteria Species 0.000 description 14
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical group OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 14
- 235000001014 amino acid Nutrition 0.000 description 8
- 229940024606 amino acid Drugs 0.000 description 8
- 235000004400 serine Nutrition 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 6
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical class OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- 108091092724 Noncoding DNA Proteins 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 125000000254 aspartoyl group Chemical group 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000012268 genome sequencing Methods 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- 235000003969 glutathione Nutrition 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000012434 nucleophilic reagent Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 102220023257 rs387907546 Human genes 0.000 description 2
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- -1 50 μ l Substances 0.000 description 1
- DVJSJDDYCYSMFR-ZKWXMUAHSA-N Ala-Ile-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O DVJSJDDYCYSMFR-ZKWXMUAHSA-N 0.000 description 1
- GHODABZPVZMWCE-FXQIFTODSA-N Asp-Glu-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O GHODABZPVZMWCE-FXQIFTODSA-N 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 244000241257 Cucumis melo Species 0.000 description 1
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 241000186394 Eubacterium Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- GDOZQTNZPCUARW-YFKPBYRVSA-N Gly-Gly-Glu Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O GDOZQTNZPCUARW-YFKPBYRVSA-N 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- VGSPNSSCMOHRRR-BJDJZHNGSA-N Ile-Ser-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N VGSPNSSCMOHRRR-BJDJZHNGSA-N 0.000 description 1
- NJGXXYLPDMMFJB-XUXIUFHCSA-N Ile-Val-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N NJGXXYLPDMMFJB-XUXIUFHCSA-N 0.000 description 1
- 108091029795 Intergenic region Proteins 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- FKQPWMZLIIATBA-AJNGGQMLSA-N Leu-Lys-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FKQPWMZLIIATBA-AJNGGQMLSA-N 0.000 description 1
- VDIARPPNADFEAV-WEDXCCLWSA-N Leu-Thr-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O VDIARPPNADFEAV-WEDXCCLWSA-N 0.000 description 1
- MPOHDJKRBLVGCT-CIUDSAMLSA-N Lys-Ala-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCCN)N MPOHDJKRBLVGCT-CIUDSAMLSA-N 0.000 description 1
- HAQLBBVZAGMESV-IHRRRGAJSA-N Met-Lys-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O HAQLBBVZAGMESV-IHRRRGAJSA-N 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 102000057361 Pseudogenes Human genes 0.000 description 1
- 108091008109 Pseudogenes Proteins 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 241000186339 Thermoanaerobacter Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000009604 anaerobic growth Effects 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-L aspartate group Chemical group N[C@@H](CC(=O)[O-])C(=O)[O-] CKLJMWTZIZZHCS-REOHCLBHSA-L 0.000 description 1
- 229960005261 aspartic acid Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 102220369447 c.1352G>A Human genes 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 238000001818 capillary gel electrophoresis Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 1
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000012177 large-scale sequencing Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 238000003754 machining Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 102220023258 rs387907548 Human genes 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 150000003355 serines Chemical class 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- FRGKKTITADJNOE-UHFFFAOYSA-N sulfanyloxyethane Chemical compound CCOS FRGKKTITADJNOE-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Abstract
The invention discloses a high temperature resistant peptidase E gene, a coded polypeptide and a preparation method thereof. It relates to a method for coding separated DNA with activity or its functional equivalent variant and utilizing recombinant DNA technique to produce polypeptide with high-temp. resistant peptidase E activity or its functional equivalent variant by using the described separated DNA. Based on sequencing and analysis of Tengchong thermophilic anaerobe whole genome, the high temperature resistant peptidase E gene is cloned and separated. The gene is useful for preparing transgenic microorganisms or animals and plants for producing high-temperature resistant peptidase E and recovering the enzyme coded by the gene. In addition, the invention also provides an amino acid sequence and a functional equivalent of the polypeptide with the high-temperature resistant peptidase E activity. Meanwhile, the invention also provides a method for preparing, separating and purifying the polypeptide with the high-temperature resistant peptidase E activity.
Description
Technical field
The present invention relates to sudden change or genetic engineering, relate in particular to a kind of refractory peptidase E gene and encoded polypeptides and preparation method.
Background technology
PEPE (PepE) is a kind of two peptide chains of N-terminal aspartate characteristic.PEPE is not suppressed by any typical peptidase inhibitors, and its amino-acid sequence does not belong to any known peptide enzymatic structure.Compare by aminoacid sequence and a series of associated protein enzyme sequence with PEPE, can define the amino-acid residue in this family, PEPE is a kind of serine hydrolase.
The sudden change of using the location to instruct forms and changes all known Serines, Histidine, and asparagicacid residue, and have been found that Serine 120 in this activating reaction, Histidine 157, and aspartic acid 135 is essential.Serine 120 is converted to halfcystine, except the activity that can survey, can also cause the reduction (10000 times) of amplitude, and this activity is not suppressed by the thiol reactant thing; This residue of these results verifications is likely the nucleophilic reagent of katalysis, simultaneously can think that PEPE is the prototype of the new family of a Serine peptase. because representative is found in Eubacteria, in a kind of insect (Drosophila) and a kind of vertebrates, rather than in the archeobacteria or in gene sequencing in available any other eukaryote, so the phylogenetic distribution of family is unusual (African melon toad belongs to).
The peptide lytic enzyme is used to proteinicly be degraded to one amino acid fully, and can be used as the source of nutrition of peptide.PEPE has the ability of hydrolysis aspartic acid-X peptide, can also specialization aspartoyl peptase.It can not hydrolysis L-glutamic acid-X, aspartoyl-X, glycine-X, or any other determined non-aspartoyl peptide.PEPE needs a peptide in N ' end and indiscrete N-zone freely.
The mechanism of action of relevant PEPE or specific general architecture basics are still under study for action.PEPE is insensitive to the inhibitor of main kind, and because order is imprecise, it does not belong to any known protein enzyme class.PEPE, different with the peptase of a lot of Salmonellass, lack any significantly can be with the metlbond thing site of its classification.Studies show that PEPE may be confirmed as a kind of novel peptide lytic enzyme.Under from the known situation of the sequence of other biological body, the PEPE homolog is identified.These sequences have demonstrated several high conserved regions territory, and wherein a kind of Gly-X-Ser-X-Gly collection of illustrative plates that comprises a typical serine hydrolase demonstrates PEPE and can utilize serine residue as nucleophilic reagent.Relevant PEPE is Serine peptase and the hypothesis that can be used as catalyzer when identifying amino acid whose residue, can form experiment by the sudden change that the site is instructed and measure.
The aminoacid sequence of PEPE is different from any known protein matter.Comprise the starter in AMP susceptor protein (CRP) site of one-period from the nucleotide sequence of the upstream region of PEPE, growing carrier and the influence of crp sudden change on the synthetic phraseology of PepE-lacZ show that PEPE is a kind of CRP regulon.PEPE shows that with respect to the shortage of the similar sequences of the specificity of any other peptase and its uniqueness this kind of enzyme may be a novel peptase prototype.
Tengchong thermophilc anaerobe (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 found by Microbe Inst., Chinese Academy of Sciences and has carried out the analysis on the taxonomy.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 refractory peptidase E that is had in its body also has its specific structure of controlling oneself.
Summary of the invention
One of purpose of the present invention provides a kind of isolating, and coding has the nucleotide sequence of the active polypeptide of refractory peptidase E.
Two of purpose of the present invention provides a kind of isolating refractory peptidase E active polypeptide that has.
Another object of the present invention also provide thermophilc anaerobe the PEPE recombinant vectors, contain the host cell of recombinant vectors, and prepare proteic method.
One aspect of the present invention provides a kind of can the coding to have the nucleotide sequence of the active polypeptide of refractory peptidase E.Said nucleotide sequence coded the have polypeptide of the aminoacid sequence among the SEQ ID NO.2 or the modified forms of described polypeptide, on this modified forms function quite or relevant with PEPE.Nucleotide sequence has the polynucleotide sequence of SEQ ID NO.1 and its mutant form, and mutation type comprises: disappearance, nonsense, insertion, missense.
The present invention provides a kind of refractory peptidase E active polypeptide on the other hand.This polypeptide has polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of the aminoacid sequence among the SEQ IDNo.2.
The present invention also provides a kind of method for preparing refractory peptidase E, and it may further comprise the steps:
1) isolate the coding refractory peptidase E nucleotide sequence SEQ ID NO.1;
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, formation can be produced the reconstitution cell of refractory peptidase E;
4) culturing step 3) in reconstitution cell;
5) separation, purifying obtain refractory peptidase E.
The present invention relates to the separation and the expression of the refractory peptidase E gene of thermophilc anaerobe.Based on Tengchong thermophilc anaerobe genome sequencing and analysis, clone and separate refractory peptidase E gene.The refractory peptidase E of transgenic microorganism or animals and plants this gene is used to produce to(for) preparation, and it is useful to reclaim the enzyme that obtains this genes encoding.In addition, the present invention also provides and has had active amino acid sequence of polypeptide of refractory peptidase E and functional equivalent body.Simultaneously, the present invention also provides preparation, separates, and purifying has the method for the active polypeptide of refractory peptidase E.
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
The invention provides isolating, the polynucleotide molecule of the active polypeptide of coding refractory peptidase E, this nucleic acid molecule is by obtaining Tengchong thermophilc anaerobe genome sequencing and analysis, nucleotide sequence with SEQ.ID NO.1, its coding has 242 amino acid whose polypeptide, and this polypeptide infers that molecular weight is 27558 dalton.
The present invention also provides a kind of recombinant vectors, and 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 the method for producing refractory peptidase E with the recombined engineering technology.
The present invention provides a kind of isolating refractory peptidase E or polypeptide further, and it has the SEQ.IDNO.2 aminoacid sequence, 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, " refractory peptidase E gene " refers to encode and has the nucleotide sequence of the active polypeptide of refractory peptidase E, as nucleotide sequence and the degenerate sequence thereof of 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 SEQ ID NO.2 of also encoding out with SEQ ID NO.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 SEQ ID NO.1.This term also comprises and 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, " refractory peptidase E " refers to have the active SEQ ID of refractory peptidase E NO.2 polypeptide of sequence.This term also comprises the varient of SEQ ID NO.2 sequence, and these varients have and natural refractory peptidase E 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 refractory peptidase E.
In the present invention, can select various carrier known in the art for use, as commercially available various plasmids, clay, phage and retrovirus etc.When producing refractory peptidase E of the present invention, the refractory peptidase E gene sequence can be linked to each other with expression regulation sequence, thereby form the refractory peptidase E expression vector.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 well known to a person skilled in the art recombinant DNA technology preparation, as can be with reference to people such as Sambrook, and 1989 or people such as Ausubel, 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.Refractory peptidase E gene full length sequence of the present invention or its fragment can be used polymerase chain reaction (PCR) 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.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the 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 (NewYork: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 in the process of finishing figure (finishing), and can solve the difficulty that cause filling-up hole in bigger hole (gap).Build storehouse flow process such as (see figure 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 PE 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: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.
Embodiment 4: sequence assembly
So-called sequence assembly is exactly full genome shotgun approach, and the sample sequence that claims the shotgun random sequencing to obtain again is assembled into successive contig (contig), 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 (Waterman MS, 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.
Embodiment 5: gene annotation
After obtaining genomic most of sequence (frame diagram of finishing the work) substantially, just need carry out note to genome, comprise out frame frame (Open Reading Frame, prediction ORF), 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 protein database) of NCBI 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 open in the frame when a plurality of promotor occurring at one, 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 pseudogenes), 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 termination is regional opens frame and 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.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 it 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 the 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 the terminal Walking of extension test
According to contig direction and position relation that A and B provided, 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 6: the preparation of PEPE and purification
According to the PEPE complete encoding sequence (SEQ ID NO.1) that gene annotation among the embodiment obtains, design can amplify the primer that complete coding is read frame, and introduces restriction endonuclease sites respectively on positive anti-primer, 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 obtains containing the engineering bacteria DH5 α-pGEX-2T-PepE of expression vector.
The engineering bacteria DH5 α-pGEX-2T-PepE 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 glutathione S epharose 4B of 20ml PBS by every 400ml bacterium, 37 ℃ of joltings were in conjunction with 30 minutes, 10000rpm precipitated the glutathione S epharose 4B that combines desirable proteins in centrifugal 10 minutes, abandoned supernatant.Add 100 μ l reduced glutathion 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 the SDS-PAGE electrophoresis, detects purification effect.Protein band at 27558 dalton place is PEPE.
1. SEQ ID NO.1 ( 1 ) :a.:729b.:DNAc.:d.: ( 2 ) : ( 3 ) :atgaaaaaagttgttgccatagggggaggagagatttcaaaatttgaaactttgaaaatagatgaagaaatagttaaactaactggtaaagccaatccaaaagctctttttataccaactgcgagcggagaacctgaaggttactgtgaaagttttgaccttgtctatggtaaaacgcttggatgcaagacagatgttttgtttttaataaaagatagtttggataggacgctaatagaagataaaatcttgtgggcagatttgatttatgttggcggtggtaacacaaagaggatgatggaaatatggaggtccaaaggtgttgacaagatgttgattgatgcttatcagagaggaacggtactttcaggattgagtgcaggagctatatgctggttcaaatacggttttagtgattcactaagatttcaggatgtagaaacatggagttatattaaagttgagggtttggggctttttgatgccatactatgtcctcatctcgacgaagaaaatagaagggaatattttgaaaaatttatgatgaactttgatggtacaggaataggattagagaatggctgtgcaattgaaatcatagaagatacttttagaattattaagtcttttgaaaatgctcatgcctatgtatttaaaagaagaagagataaattgtttgttgaagagataaagaatgacgattaccttccatttaatttgctaagttag2. SEQ ID NO.2 ( 1 ) :a.:242b.:c.:d.: ( 2 ) : ( 3 ) Met Lys Lys Val Val Ala Ile Gly Gly Gly Glu Ile Ser Lys Phe Glu1 5 10 15Thr Leu Lys Ile Asp Glu Glu Ile Val Lys Leu Thr Gly Lys Ala Asn
20 25 30Pro?Lys?Ala?Leu?Phe?Ile?Pro?Thr?Ala?Ser?Gly?Glu?Pro?Glu?Gly?Tyr
35 40 45Cys?Glu?Ser?Phe?Asp?Leu?Val?Tyr?Gly?Lys?Thr?Leu?Gly?Cys?Lys?Thr
50 55 60Asp?Val?Leu?Phe?Leu?Ile?Lys?Asp?Ser?Leu?Asp?Arg?Thr?Leu?Ile?Glu65 70 75 80Asp?Lys?Ile?Leu?Trp?Ala?Asp?Leu?Ile?Tyr?Val?Gly?Gly?Gly?Asn?Thr
85 90 95Lys?Arg?Met?Met?Glu?Ile?Trp?Arg?Ser?Lys?Gly?Val?Asp?Lys?Met?Leu
100 105 110Ile?Asp?Ala?Tyr?Gln?Arg?Gly?Thr?Val?Leu?Ser?Gly?Leu?Ser?Ala?Gly
115 120 125Ala?Ile?Cys?Trp?Phe?Lys?Tyr?Gly?Phe?Ser?Asp?Ser?Leu?Arg?Phe?Gln
130 135 140Asp?Val?Glu?Thr?Trp?Ser?Tyr?Ile?Lys?Val?Glu?Gly?Leu?Gly?Leu?Phe145 150 155 160Asp?Ala?Ile?Leu?Cys?Pro?His?Leu?Asp?Glu?Glu?Asn?Arg?Arg?Glu?Tyr
165 170 175Phe?Glu?Lys?Phe?Met?Met?Asn?Phe?Asp?Gly?Thr?Gly?Ile?Gly?Leu?Glu
180 185 190Asn?Gly?Cys?Ala?Ile?Glu?Ile?Ile?Glu?Asp?Thr?Phe?Arg?Ile?Ile?Lys
195 200 205Ser?Phe?Glu?Asn?Ala?His?Ala?Tyr?Val?Phe?Lys?Arg?Arg?Arg?Asp?Lys
210 215 220Leu?Phe?Val?Glu?Glu?Ile?Lys?Asn?Asp?Asp?Tyr?Leu?Pro?Phe?Asn?Leu225 230 235 240Leu?Ser
Claims (8)
1. isolated DNA molecule is characterized in that: it is the nucleotide sequence that coding has the polypeptide of refractory peptidase E protein-active.
2. dna molecular as claimed in claim 1 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 described polypeptide, on this modified forms function quite or relevant with refractory peptidase E.
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 the refractory peptidase E 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. method for preparing refractory peptidase E is characterized in that this method may further comprise the steps:
1) isolate the coding refractory peptidase E gene nucleotide sequence SEQ ID NO.1;
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, formation can be produced the reconstitution cell of refractory peptidase E;
4) culturing step 3) in reconstitution cell;
5) separation, purifying obtain refractory peptidase E.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 02110735 CN1379093A (en) | 2002-02-01 | 2002-02-01 | High-temperature resistant peptidase E gene and its coded polypeptide and preparing method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 02110735 CN1379093A (en) | 2002-02-01 | 2002-02-01 | High-temperature resistant peptidase E gene and its coded polypeptide and preparing method |
Publications (1)
Publication Number | Publication Date |
---|---|
CN1379093A true CN1379093A (en) | 2002-11-13 |
Family
ID=4741215
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN 02110735 Pending CN1379093A (en) | 2002-02-01 | 2002-02-01 | High-temperature resistant peptidase E gene and its coded polypeptide and preparing method |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN1379093A (en) |
-
2002
- 2002-02-01 CN CN 02110735 patent/CN1379093A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN1418954A (en) | High temp. resistant HSP 70 molecular chaperone and coded polypeptide and preparation process thereof | |
CN1420172A (en) | High-temp resistant CTP synthetase gene, polypeptide coded therewith and preparing method thereof | |
CN1379093A (en) | High-temperature resistant peptidase E gene and its coded polypeptide and preparing method | |
CN1198928C (en) | High-temperature resistant ribosomal protein L15 gene and its coded polypeptide and preparing process | |
CN1164750C (en) | High-temperature resistant transaldolase gene, polypeptide coded by same and preparation method of polypeptide | |
CN1371996A (en) | High-temperature resistant spermidine synthase gene sequence, polypeptide coded by same and method for preparing polypeptide | |
CN1417338A (en) | High temperature-resisting DNA polymerase gene sequence and its encoded polypeptide and prepn process | |
CN1379094A (en) | High-temperature-resistant tyrosyl tRNA synthetase gene, polypeptide coded by same and preparation method | |
CN1366059A (en) | Refractory phosphomannose isomerase gene and its polypeptide coded by it and preparing process | |
CN1379103A (en) | High-temperature resistant glucan phosphorylase gene and polypeptide coded by same and preparation method | |
CN1379101A (en) | Refractory thymidine phosphorylase gene and its polypeptide coded by it and preparing process | |
CN1390943A (en) | High-temp. resistant 6-phosphoglucose isomerase gene and its coded polypeptide and preparing process | |
CN1371998A (en) | High-temp. resistant guanosine triphosphate cyclic hydrolase gene sequence and its coded polypeptide and preparation method | |
CN1379100A (en) | High-temp. resistant dihydroorotate synthase gene and its coded polypeptide and preparing method | |
CN1366054A (en) | Refractory glutaminic acid imine methyltransferase gene and its polypeptide coded by it and preparing process | |
CN1420173A (en) | High-temp. resistant biotin synthetase gene, polypeptide coded therewith and preparing method thereof | |
CN1367249A (en) | High-temp. resistant chorismate synthase gene and its coded polypeptide and preparation method | |
CN1379095A (en) | High-temp. resistant exo-polyphosphate enzyme gene and its coded polypeptide and preparing process | |
CN1382804A (en) | High-temperature-resistant phosphoribosyl anthranilate isomerase gene, polypeptide coded by same and preparation method of polypeptide | |
CN1379107A (en) | High-temp. resistant cysteine synthase gene and its coded polypeptide and preparing method | |
CN1418955A (en) | High temp. resistant FtsA protein gene and coded polypeptide and preparation process thereof | |
CN1420175A (en) | High-tamp. resistant threonine synthetase gene, polypeptide coded therewith and preparing method thereof | |
CN1418958A (en) | High temp. resistant urocanate hydratase gene and coded polypeptide and preparation proess thereof | |
CN1379108A (en) | Refractory phosphoheptose isomerase gene and its polypeptide coded by it and preparing process | |
CN1379096A (en) | High-temperature resistant histidyl tRNA synthetase gene and polypeptide coded by same and preparation method |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
WD01 | Invention patent application deemed withdrawn after publication |