CN103468656A - Purine nucleoside phosphorylase and preparation method thereof - Google Patents
Purine nucleoside phosphorylase and preparation method thereof Download PDFInfo
- Publication number
- CN103468656A CN103468656A CN2013104513238A CN201310451323A CN103468656A CN 103468656 A CN103468656 A CN 103468656A CN 2013104513238 A CN2013104513238 A CN 2013104513238A CN 201310451323 A CN201310451323 A CN 201310451323A CN 103468656 A CN103468656 A CN 103468656A
- Authority
- CN
- China
- Prior art keywords
- purine nucleoside
- nucleoside phosphorylase
- sudden change
- host cell
- polynucleotide
- 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.)
- Granted
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
The invention relates to purine nucleoside phosphorylase and a preparation method of the purine nucleoside phosphorylase, in particular to a mutational pseudoalteromonas-genus purine nucleoside phosphorylase. Compared with a wild-type amino acid sequence, the amino acid sequence of the mutational purine nucleoside phosphorylase is characterized in that Asp at the 97th bit is mutated into Tyr. The invention further discloses the preparation method of the purine nucleoside phosphorylase. The purine nucleoside phosphorylase and the preparation method of the purine nucleoside phosphorylase have the advantages of being high in yield and good in heat stability. The invention further discloses polynucleotide for encoding the amino acid sequence of the purine nucleoside phosphorylase, a carrier containing the polynucleotide and a host cell containing the polynucleotide.
Description
Technical field
The present invention relates to the genetically engineered field, relate to particularly a kind of purine nucleoside phosphorylase and preparation method thereof.
Background technology
Purine nucleoside phosphorylase (purine nucleoside phosphorylase), be called for short PNP, is one of key enzyme of Purine salvage pathway, extensively is present in Mammals, parasite and microorganism.Protein structure according to purine nucleoside phosphorylase can be divided into two classes: the homo-hexamer class of low-molecular-weight homotrimer class and high molecular.Mammals and part microorganism (Salmonella typhimurium salmonella typhimurium for example wherein, hyperthermophilic archaeon strain sesame field sulfolobus solfataricus Sulfolobus solfataricus etc.) purine nucleoside phosphorylase belongs to the homotrimer class, its molecular weight is about 80～100kDa, the molecular weight of each subunit is 30～32kDa, and this type of purine nucleoside phosphorylase can only be usingd guanosine-and inosine as substrate usually.And the purine nucleoside phosphorylase of homo-hexamer class, Substratspezifitaet is not strong, both can accept guanosine-and inosine as substrate, also can adenosine as substrate.Its molecular weight is about 150kDa, and molecular weight subunit is the 25kDa left and right.
Due to the special biological activity of PNP, make it at field of medicaments, obtain larger application: 1. be applied to the synthetic of nucleoside medicine, quantity research shows greatly, and ucleosides (nucleosides and analogue thereof) has antitumor antiviral effect widely.2. be applied to neoplasm targeted therapy; 3. be applied to the quantitative assay of inorganic phosphorus and ATPase enzyme activity etc.
In view of the wide application prospect of PNP, in recent years, more and more about the genetically engineered research of PNP, but be main mainly with intestinal bacteria PNP and thermophile bacteria PNP.And the present invention chooses the PNP of clone's Pseudoalteromonas (Pseudoalteromonas), referred to as PiPNP, its PNP with respect to other Pseudomonas has a significant advantage, this enzyme has a higher catalytic efficiency under low temperature (being less than 40 ℃), research is found, in vitro, when the inosine of catalysis lower concentration or high density reacts, the optimum temperuture of PiPNP is 30-35 ℃ or 50-60 ℃, the PNP(that is starkly lower than other Pseudomonas for example inosine reaction optimum temperuture of intestinal bacteria PNP catalysis lower concentration or high density is respectively 50 ℃ or 60-70 ℃), this just makes at the popular response temperature (37 ℃), PiPNP is used less amount just can obtain higher catalytic efficiency.But PiPNP also exists compared to the PNP of other Pseudomonas, its defect---temperature stability is lower, PiPNP is incubated to 30min, and in the time of 37-42 ℃, enzyme is lived and is occurred sharply to change, 50 ℃ of complete deactivations, EcPNP sharply changes 50-55 ℃ of generation, completely loses inactivation in the time of 65 ℃.
In addition, according to the each side research of having reported, the PNP output of recombinant bacterium fermentative production is still lower, mostly is 100～200U/ml, for suitability for industrialized production, still can not reach good effect, and output has much room for improvement.
Summary of the invention
The purine nucleoside phosphorylase that the object of the present invention is to provide the molecular modification of a kind of process and there is high thermal stability, and the method for this purine nucleoside phosphorylase of preparation of high yield.
First aspect present invention provides a kind of purine nucleoside phosphorylase of Pseudoalteromonas of sudden change, with the aminoacid sequence of wild-type, compares, and in the aminoacid sequence of the purine nucleoside phosphorylase of described sudden change, the 97th Asp sports Tyr.
In another preference, the purine nucleoside phosphorylase of described sudden change has following one or more characteristics:
(a), than enzyme work >=30U/mg, be preferably >=45U/mg;
(b) be stored in alkaline environment;
(c) deactivation temperature >=60 ℃;
(d) at 0-55 ℃, shelf time >=3h.
In another preference, the optimum pH of the purine nucleoside phosphorylase of described sudden change is 7.0-9.0, is preferably 7.5-8.5, is more preferably 8.0.
In another preference, its aminoacid sequence is as shown in SEQ ID NO.1.
Second aspect present invention provides a kind of polynucleotide of separation, the purine nucleoside phosphorylase of the described sudden change of described polynucleotide encoding first aspect.
In another preference, the one or more codons in described polynucleotide sequence in codon AGG, CTA, CTA, ATA, CTA and the CTA of coded amino acid Arg2, Leu24, Leu77, Ile87, Leu134 and Leu145 are replaced by the corresponding preference codon of intestinal bacteria.
In another preference, the one or more codons in described polynucleotide sequence in codon AGG, CTA, CTA, ATA, CTA and the CTA of coded amino acid Arg2, Leu24, Leu77, Ile87, Leu134 and Leu145 are replaced by CGA, CTG, CTG, ATC, CTG and CTG.
In another preference, described polynucleotide sequence is as shown in SEQ ID NO.2.
Third aspect present invention provides a kind of carrier, and described carrier contains the described polynucleotide of second aspect.
In another preference, described carrier is expression vector, more preferably for being adapted at the expression vector of expression in escherichia coli.
Fourth aspect present invention provides a kind of host cell, it is characterized in that, described host cell comprises in the described carrier of the third aspect or genome and is integrated with the described polynucleotide of second aspect.
In another preference, described host cell is prokaryotic cell prokaryocyte or eukaryotic cell.
In another preference, described host cell is intestinal bacteria (E.coli) cells.
Fifth aspect present invention provides the preparation method of the purine nucleoside phosphorylase of the described sudden change of a kind of first aspect, comprises the following steps:
(a) under conditions suitable for the expression, cultivate the described host cell of fourth aspect, thereby give expression to the purine nucleoside phosphorylase of the described sudden change of first aspect;
(b) separate the purine nucleoside phosphorylase of described sudden change.
In another preference, described host cell is intestinal bacteria.
In another preference, in step (a) before, described method is further comprising the steps of:
(1) provide the polynucleotide sequence of the purine nucleoside phosphorylase of a Pseudoalteromonas;
(2) by the codon that is coding Tyr corresponding to the codon mutation of the 97th Asp in the aminoacid sequence of the purine nucleoside phosphorylase of wild-type Pseudoalteromonas in described polynucleotide sequence, and optionally, by coded amino acid Arg2 in described polynucleotide sequence, Leu24, Leu77, Ile87, one or more codon AGG in Leu134 and Leu145, CTA, CTA, ATA, CTA replaces with the corresponding preference codon of intestinal bacteria with CTA, thereby make the polynucleotide sequence of the purine nucleoside phosphorylase of the described sudden change of coding first aspect,
(3) described polynucleotide sequence previous step made imports host cell, thereby makes the described host cell of fourth aspect.
In another preference, the polynucleotide sequence in step (1) is as shown in SEQ ID NO.3.
In another preference, the enzyme of described wild-type or wild-type is the purine nucleoside phosphorylase from Pseudoalteromonas (Pseudoalteromonas).
In another preference, the codon GAC of the 97th Asp is sported to the codon TAT of coding Tyr.
In another preference, described codon AGG, CTA, CTA, ATA, CTA and CTA are replaced with to CGA, CTG, CTG, ATC, CTG and CTG.
In another preference, the expression activity >=300U/ml of the purine nucleoside phosphorylase of the sudden change that described method obtains, be preferably 350-450U/ml.
Sixth aspect present invention provides a kind of method of transforming purine nucleoside phosphorylase, and described method comprises step:
(1) provide the polynucleotide sequence of the purine nucleoside phosphorylase of a Pseudoalteromonas;
(2) by the codon that is coding Tyr corresponding to the codon mutation of the 97th Asp in the aminoacid sequence of the purine nucleoside phosphorylase of wild-type Pseudoalteromonas in described polynucleotide sequence, thereby make the polynucleotide sequence of the purine nucleoside phosphorylase of encoding mutant;
(3) express the described polynucleotide sequence that previous step makes, thereby make the purine nucleoside phosphorylase of sudden change.
In another preference, described method is for improving the stability of purine nucleoside phosphorylase.
In another preference, the polynucleotide sequence in step (1) is as shown in SEQ ID NO.3.
Seventh aspect present invention provides the purposes of the purine nucleoside phosphorylase of the described sudden change of a kind of first aspect, it is characterized in that, for the catalysis inosine, carries out the phosphorolysis reaction; Or with making the catalyzer that inosine is carried out to the phosphorolysis reaction.
Eighth aspect present invention provides a kind of catalyzed reaction, comprise step: under the purine nucleoside phosphorylase or the described host cell existence of fourth aspect of the described sudden change of first aspect, inosine is carried out to enzymic catalytic reaction (phosphorolysis reaction), generate xanthoglobulin.
Ninth aspect present invention provides a kind of zymin, the purine nucleoside phosphorylase that described zymin contains the described sudden change of first aspect.
In another preference, described zymin is immobilized enzyme.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can combining mutually between specifically described each technical characterictic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tire out and state no longer one by one at this.
The accompanying drawing explanation
The plasmid map that Fig. 1 is PNP (O)-pET-28a (+).
The purpose band electrophorogram that Fig. 2 A is pcr amplification.
Fig. 2 B is PNP (O)-pET-28a (+) screening positive clone electrophorogram
The structural representation that Fig. 3 is the pMD19-T carrier.
Fig. 4 is codon optimized engineering bacteria PNP (O)-pET-28a (+)-BL21 and the engineering bacteria PNP-pET-28a (+) do not optimized-BL21 fermented liquid SDS-PAGE figure.
Fig. 5 is codon optimized engineering bacteria PNP (O)-pET-28a (+)-BL21 and the secreting, expressing yield comparison data plot of the engineering bacteria PNP-pET-28a (+) do not optimized-BL21.
Fig. 6 is for changing the codon of Ile87 into ATT and changing the correlation data figure of expression activity between the engineering bacteria of ATC into.
The enzyme reservation vigor that Fig. 7 is PiPNP and rPiPNP varies with temperature figure.
The enzyme that Fig. 8 is PiPNP and rPiPNP retains vigor temporal evolution figure.
Embodiment
The contriver is through research extensively and profoundly, be surprised to find that, by changing the codon TAT of coding Tyr into from the codon GAC of coding Asp97 in the purine nucleoside phosphorylase PNP gene order of Pseudoalteromonas (Pseudoalteromonas), prepared mutant enzyme has the thermostability of obvious raising.In addition, by by after in the PNP gene order, codon AGG, CTA, CTA, ATA, CTA and the CTA of coded amino acid Arg2, Leu24, Leu77, Ile87, Leu134 and Leu145 change CGA, CTG, CTG, ATC, CTG and CTG into respectively, can further significantly improve the output of PNP through gene engineering preparation method.Completed on this basis the present invention.
As used herein, " separation " refers to that material separates (if natural substance, primal environment is natural surroundings) from its primal environment.As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, for separation and purification.
Polynucleotide of the present invention can be DNA form or rna form.DNA form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.
The invention still further relates to the varient of above-mentioned polynucleotide, it is encoded protein fragments, analogue and the derivative of identical aminoacid sequence with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural occurs.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing in fact the function of its code book invention enzyme.
As used herein, term " primer " refer to template pairing, can take under the effect of archaeal dna polymerase that it synthesizes for starting point and the general name of the Nucleotide of living alone as a widow of the DNA chain of template complementation.Primer can be natural RNA, DNA, can be also any type of natural nucleotide.Primer can be even that non-natural Nucleotide is as LNA or ZNA etc.A special sequence complementation on primer " haply " (or " basically ") and template on chain.Primer must with template on an abundant complementation of chain could start to extend, but the sequence of primer needn't with the sequence complete complementary of template.Such as, add the preceding paragraph and the not complementary sequence of template at the 5' end of 3' end and the primer of template complementation, such primer still haply with the template complementation.As long as have sufficiently long primer can with the sufficient combination of template, the primer of non-complete complementary also can form primer-template composite with template, thereby is increased.
The Nucleotide full length sequence of enzyme of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be according to published relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually need to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplified is stitched together by proper order.
Once obtain relevant sequence, just can obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier by it, then proceeds to cell, then by ordinary method, from the host cell propagation, separates and obtains relevant sequence.
In addition, also can synthesize relevant sequence, especially fragment length more in short-term by the method for synthetic.Usually, by first synthetic a plurality of small segments, and then connect and can obtain the fragment that sequence is very long.
The method of application round pcr DNA amplification/RNA is optimized for and obtains gene of the present invention.Primer for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
The present invention also relates to the carrier that comprises polynucleotide of the present invention, and the host cell produced through genetically engineered with carrier of the present invention or albumen coded sequence, and the method that produces enzyme of the present invention through recombinant technology.
By conventional recombinant DNA technology, can utilize polynucleotide sequence of the present invention to express or produce purine nucleoside phosphorylase.In general following steps are arranged:
(1). with the polynucleotide (or varient) of code book invention albumen of the present invention, or transform or transduction appropriate host cell with the recombinant expression vector that contains these polynucleotide;
(2). cultivate host cell in suitable medium;
(3). separation, purifying purine nucleoside phosphorylase from substratum or cell.
Method well-known to those having ordinary skill in the art can be for building containing the DNA sequences encoding of enzyme of the present invention and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described DNA sequence dna can be effectively connected on the suitable promotor in expression vector, synthetic to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, phenotypic character with the host cell that is provided for select transforming, as eukaryotic cell is cultivated Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of use, or for colibacillary tsiklomitsin or amicillin resistance.
Comprise above-mentioned suitable DNA sequence dna and the suitable carrier of promotor or control sequence, can be for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, Bacillus subtillis, the bacterial cell of streptomyces; The fungal cell is as pichia spp, brewing yeast cell; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, NS0, COS7 or 293 cells etc.Correspondingly, also can will derive from the polynucleotide sequence of purine nucleoside phosphorylase of Pseudoalteromonas the codon of coded amino acid Arg2, Leu24, Leu77, Ile87, Leu134 and Leu145 respectively and replace with the codon of above-mentioned host cell preference.
With the recombinant DNA transformed host cell, can carry out with routine techniques well known to those skilled in the art.When the host is prokaryotic organism during as intestinal bacteria, the competent cell that can absorb DNA can, in exponential growth after date results, be used CaCl
2method is processed, and step used is well-known in this area.Another kind method is to use MgCl
2.If necessary, transforming the also method of available electroporation carries out.When the host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, the conventional mechanical method is as microinjection, electroporation, liposome packing etc.
The transformant obtained can be cultivated by ordinary method, expresses the protein of coded by said gene of the present invention.According to host cell used, substratum used in cultivation can be selected from various conventional mediums.Under the condition that is suitable for the host cell growth, cultivated.After host cell grows into suitable cell density, induce the promotor of selection by suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
Extracellular can be expressed or be secreted into to protein in the above methods in cell or on cytolemma.If necessary, can utilize its physics, chemical separating and purifying protein by various separation methods with other characteristic.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation processes, process the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods with protein precipitant.
The present invention includes following major advantage:
(1) purine nucleoside phosphorylase of the present invention has high thermal stability, and the enzyme deactivation temperature can be increased to more than 60 ℃, and at 0-55 ℃, more than the shelf time of enzyme extends to 3h.
(2), when purine nucleoside phosphorylase thermostability of the present invention improves, also keep higher enzymic activity.
(3) preparation method of the present invention has greatly improved the output of purine nucleoside phosphorylase, and the expression activity of product can meet or exceed 400U/mL, and this is that current gene engineering preparation method can't be reached.Thereby be conducive to large-scale industrial production.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can arbitrary combination.All features that this case specification sheets discloses can with any composition forms use, each feature disclosed in specification sheets, the alternative characteristics that can anyly be provided identical, impartial or similar purpose replaces.Therefore except special instruction is arranged, the feature disclosed is only the general example of equalization or similar features.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, such as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber are weight percent and parts by weight.
Unless otherwise defined, the same meaning that all specialties of using in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.
The source of the experiment material in the embodiment of the present invention is as follows:
PET-28a (+) is purchased from Novagen company
PMD19-T Simple Vector is purchased from TaKaRa company
Solution I is purchased from TaKaRa company
The T4DNA ligase enzyme is purchased from TaKaRa company
10*T4DNA ligase enzyme damping fluid is purchased from TaKaRa company
Tryptones is purchased from source, Shanghai consor thing Science and Technology Ltd.
Yeast powder is purchased from Angel Yeast Co.,Ltd
NaCl is purchased from Chemical Reagent Co., Ltd., Sinopharm Group
The optimization of embodiment 1 Pseudoalteromonas (Pseudoalteromonas) PNP gene order and the structure of cloning vector
1. the optimization of gene order
The optimization of gene order is by the PNP gene order of Pseudoalteromonas sp.i590 (GEN BANK EF222283, SEQ ID NO.3) carry out the part molecular modification, be divided into two: the one, change codon AGG, CTA, CTA, ATA, CTA, the CTA of the Arg2 of the coded amino acid in the whole piece sequence, Leu24, Leu77, Ile87, Leu134, Leu145 into CGA, CTG, CTG, ATC, CTG, CTG; The 2nd, transform 97 Asp as Tyr, codon changes TAT into by GAC.It is synthetic that gene order after optimization is carried out full gene.
Full gene is synthetic:
Entrusting Shanghai to give birth to work synthesizes.At first obtain the PNP gene order of the wild-type of Pseudoalteromonas sp.i590 from ncbi database, and the codon of the 97th amino acids residue changes Tyr into from Asp97.In addition, the inventor in order to realize high efficient expression in intestinal bacteria, gene order is optimized, wherein mainly six place's codons is replaced with to intestinal bacteria preference codon (soon six codons in codon AGG, CTA, CTA, ATA, CTA and the CTA of Arg2, Leu24, Leu77, Ile87, Leu134 and Leu145 are replaced by CGA, CTG, CTG, ATC, CTG and CTG).The complete synthesis employing synthetic method of described sequence is synthesized.
Synthetic be by will be synthetic 3 '-end of oligonucleotide chain first with 3 '-OH, with an insoluble carrier, be connected; then from 3 '-5 ' direction, nucleotide monomer is added up successively; the active function groups of the nucleotide monomer used is all through overprotection; in the time of on adding nucleotide chain to; remove the protection of 3 ' end, then discharge again 5 ' end in order to connect next nucleotide monomer.
2. the design of primer
According to the gene order after above-mentioned optimization and pET-28a(+) restriction enzyme site of (being purchased from Novagen company), utilize the Primer5.0 primer-design software to carry out the design of primer:
Upstream primer (primer 1) 5 '-ctagctagcatgcgaactccgcatatcaatg-3 ' (SEQ ID No.4)
Downstream primer (primer 2) 5 '-ccgctcgagttagatagactctaatgcaattttaac-3 (SEQ ID No.5)
3. the amplification of gene
The full gene product of chemosynthesis of take is template, carries out the PCR reaction under the catalysis of DNA Taq enzyme.Reaction system and reaction conditions are distinguished as shown in Table 1 and Table 2:
Table 1 reaction system
| Composition | Volume (ul) |
| 10×buffer（with?MgCl 2） | 5 |
| Taq?Plus? |
1 |
| |
1 |
| |
2 |
| |
2 |
| |
1 |
| ddH 2O | 38 |
| Total amount | 50ul |
Table 2 reaction conditions
The PCR product is through 1.5% agarose gel electrophoresis, with DL2000
tMdNA Marker is contrast, after checking gene fragment size is correct, reclaims test kit (Shanghai is given birth to work UNIQ-10 pillar DNA glue and reclaimed test kit) with glue and reclaims the purpose fragment, and concrete operation method can be referring to specification sheets.Electrophorogram as shown in Figure 2 A.
4. connect
The purpose fragment that recovery is obtained is connected to pMD19-TSimple Vector(T carrier) (purchased from TakaRa company, structure is as Fig. 3) upper, and linked system is as shown in table 3, and 16 ℃ of connections are spent the night.
Table 3 linked system
| Composition | Volume (ul) |
| pMD19-TSimple?Vector | 0.5μl |
| Solution I(is purchased from TaKaRa company) | 2.5μl |
| The purpose fragment | 2μl |
| Total amount | 5μl |
5. transform
The mono-bacterium colony of picking BL21 (comprises Tryptones 1.0%, yeast powder 0.5%, NaCl1.0% to 50ml LB liquid nutrient medium, adding final concentration after sterilizing is 100 μ g/ml penbritins) in 37 ℃, the 220rpm shaking culture, OD600 to 0.3～0.4, stop shaking bacterium; Add 1ml bacterium liquid in the 1.5mlEP pipe, 4 ℃, 4500rpm, centrifugal 5min, collect thalline, abandons supernatant; Add the ice-cold CaCl2 of 600ul100mM, suction is beaten and is made cell suspension gently, ice bath 30min, and 4 ℃, the centrifugal 5min of 4500rpm, abandon supernatant, collecting cell; Add the CaCl2 solution re-suspended cell that 40ul100mM is ice-cold, mix gently, put into 4 ℃ of refrigerators, use in 4～24 hours.
The EP pipe that competent cell is housed is placed on ice, at super clean bench, adds T carrier connecting fluid 5ul, mix ice bath 30min; The EP pipe is transferred in 42 ℃ of water-baths, accurately places 90s(motionless); Be transferred to rapidly ice bath, 2～3min.Add 800ul LB substratum, 37 ℃ of shaking culture 1～2 hour, to recover resistance.4 ℃, 4500rpm, 1min, abandon the 600ul supernatant, slightly mixes, by its coating LBA dull and stereotyped (comprise Tryptones 1.0%, yeast powder 0.5%, NaCl1.0%, agar 2.0%, adding final concentration after sterilizing is 100 μ g/ml penbritins).Just putting to liquid for 37 ℃ and be absorbed (about 10min).Be inverted for 37 ℃ and cultivate 15 hours, observe and have transformed bacteria to generate, flat board is put into to 4 ℃ of refrigerators.
6. bacterium colony PCR screening positive clone
The bacterium colony transformed on flat board is forwarded to the LBA flat board, is inverted for 37 ℃ and cultivates 12～16h, using the bacterium of transferring on flat board as pcr template, PCR reaction conditions, reaction system are with the 3. step.1.5% agarose gel electrophoresis check, select positive colony (called after PNP(O)-pMD19T-BL21) deliver to the order-checking of Shanghai Mei Ji biotech firm.
Embodiment 2PNP (O)-pET-28a (+)-BL21 expresses the structure of engineering bacteria
1. plasmid extraction
The correct positive colony of order-checking is inoculated in the LBA substratum, and 37 ℃, 220rpm shaking culture 12h, extract recombinant plasmid with the Plasmid Miniprep Kit of BIOMIGA, and concrete operation method is referring to specification sheets.
2. enzyme is cut
PNP (O)-pMD19T-BL21 and carrier pET-28a (+) all use Nhe I and Xho I double digestion, and 37 ℃ are spent the night; Enzyme is cut to product all for sepharose leveling board electrophoresis, and gum concentration is 1.0% (W/V), with the DL2000 of TaKaRa
tMdNA Marker is as DNA molecular amount standard; Reclaim test kit with UNIQ-10 pillar DNA glue after electrophoresis and reclaim the purpose fragment.
3. connect
Linked system is as shown in table 4, and 16 ℃ of connections are spent the night.The plasmid figure of gained recombinant vectors as shown in Figure 1.
Table 4 linked system
| Composition | Volume (ul) |
| PET-28a (+) carrier segments | 1μl |
| The goal gene fragment | 7μl |
| T4DNA Ligase(is purchased from TaKaRa company) | 1μl |
| 10*T4DNA Ligase Buffer(is purchased from TaKaRa company) | 1μl |
| ? | Totally 10 μ l |
4. transform
Concrete grammar, with embodiment 1, only replaces with kantlex by the microbiotic added in the LB substratum by penbritin and gets final product, and final concentration is 50ug/ml.
5. bacterium colony PCR screening positive clone
Concrete grammar, with embodiment 1, only replaces with by penbritin the kantlex that final concentration is 50ug/ml by the microbiotic added in the LB substratum and gets final product, positive expression clone called after PNP (O)-pET-28a (+)-BL21 of gained.The result of electrophoresis checking is as shown in Fig. 2 B, wherein the positive clone's of 1-4 electrophorogram.
The abduction delivering of embodiment 3 engineering bacterias
In picking engineering bacteria list bacterium colony access seed culture medium (containing the LB substratum of 50ug/ml kantlex), 37 ℃, the 220rpm shaking culture is to OD
600nm=0.5 left and right; Kind of daughter bacteria liquid is forwarded in new LB substratum (containing the 50ug/ml kantlex) with 2% inoculum size, and as parallel control, every bottle of seed liquor is switchable to 3 bottles of LB substratum, and 37 ℃, 220rpm shaking culture are to OD
600nm=1 left and right, the IPTG that the interpolation final concentration is 1mmol/L induces.
The mensuration of embodiment 4PNP activity
Based on take inosine as substrate, PNP can generate its phosphorolysis the enzyme activity that PNPase is measured in hypoxanthic reaction.
Preparation 200uL standard reaction liquid contains 0.5mM inosine and 25mM pH7.5 sodium phosphate buffer, add l uL enzyme liquid, 37 ℃ are reacted 10 minutes, reaction is boiled 5 minutes enzymes that go out after finishing immediately, add 100uL XOD (0.05U) after cooling, 25 ℃ are continued reaction 1 hour, detect the content that generates uric acid under the 293nm condition.The PNP enzyme activity unit is expressed as per minute catalysis and generates the enzyme amount that l umol xanthoglobulin needs.The comparing result data that table 5 and Fig. 5 are expression activity before and after gene order is optimized, table 6 and Fig. 6 are the contrast that changes the codon of Ile87 into ATT and change expression activity between the engineering bacteria of ATC into.
Table 5 gene order is optimized front and back expression activity contrast
Show 6Ile87(ATT) and Ile87(ATC) the active contrast of engineering bacterium expression
The SDS-PAGE of embodiment 5 expression products analyzes
1. the concrete composition of glue is as shown in table 7
Table 7
2. sample preparation: sample and Loading buffer(5 *) with the ratio of 4:1, mix, 100 ℃ of heated and boiled 5min make protein denaturation, respectively get the 20ul loading.
3. electrophoresis: the about 2h of electrophoresis under 100V arrives the electrophoresis chamber bottom to bromjophenol blue.
4. dye, decolour:
Gel be placed in staining fluid (50% methyl alcohol, 10% ethanol, 0.2%R-250) in, microwave treatment 30s, jog gel 30min; Gel is taken out from staining fluid, add the 100ml deionized water, washing 1min, remove moisture, adds destainer (45% methyl alcohol, 10% Glacial acetic acid) decolouring.
According to the aminoacid sequence of improved PiPNP, and 6 each Histidines that carry and the aminoacid sequence of zymoplasm restriction enzyme site, the molecular weight of expression product should be 27345.6185Da, and the SDS-PAGE result is as shown in Figure 4.
Embodiment 6 thermostability contrast experiments
1. 1.5mg/ml enzyme liquid is determined to holding temperature according to concrete experimental result respectively in 0 ℃～60 ℃ scopes, each is incubated 30 minutes, measures residual enzyme and lives.Enzyme activity at the highest vigor temperature is decided to be to 100%.The enzyme liquid of being measured has two groups, is respectively: the former sequence recombined engineering of PiPNP(group bacterium is expressed the PNP obtain), the recombinant bacterium after the optimization of rPiPNP(gene order expresses the PNP obtained), the thermostability experimental result is as shown in table 8 and Fig. 7.
Table 8PiPNP and rPiPNP are incubated enzyme contrast alive under differing temps
By data in table 8 and Fig. 7, can obviously be found out, the PiPNP of wild-type is in the time of 40～50 ℃, and enzyme is lived and sharply to be descended, complete deactivation during to 50 ℃, and improved rPiPNP is after 50 ℃, enzyme is lived and obviously decline just occurred, inactivation during to 60 ℃.
2. 1.5mg/ml enzyme liquid is incubated to 10min, 30min, 1h, 2h, 6h, 12h, 24h respectively under 4 ℃, 37 ℃ conditions, measures residual enzyme and live.The enzyme activity that does not carry out any insulation processing is decided to be to 100%.The enzyme liquid of being measured has two groups, is respectively: the former sequence recombined engineering of PiPNP(group bacterium is expressed the PNP obtain), the recombinant bacterium after the optimization of rPiPNP(gene order expresses the PNP obtained), result is as shown in table 9 and Fig. 8.
Table 9PiPNP with rPiPNP37 ℃ under the enzyme of insulation different time live to contrast
By data in table 9 and Fig. 8, can obviously be found out, the PiPNP of wild-type is under 37 ℃, and the shorter 2h of soaking time left and right residual enzyme is lived extremely low, substantially can't detect enzyme after 3h and lives, and improved rPiPNP has still retained the enzyme activity of 50% left and right in the 2h left and right, during to 3h, humble activity can also be detected.
3. the former sequence recombined engineering of the PiPNP(group bacterium taken respectively after purifying is expressed the PNP obtained) and the optimization of rPiPNP(gene order after the PNP that obtains of recombinant bacterium expression) 10mg, purity all is greater than 95%, after dissolving with the 1ml ultrapure water, measure respectively its protein content and enzyme and live.Result is as shown in table 10.
The enzyme of table 10PiPNP and rPiPNP is lived and is contrasted
Data from table 10 relatively can find out, the enzyme of improved rPiPNP is lived and do not reduced, and with the enzyme work of wild-type PiPNP, compares, and the two specific activity is suitable, even also slightly is improved.
By above-mentioned two groups of experimental results, can reach a conclusion, when the rPiPNP thermostability after molecular modification is improved significantly, also kept higher enzymic activity.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. the purine nucleoside phosphorylase of the Pseudoalteromonas of a sudden change, is characterized in that, with the aminoacid sequence of wild-type, compares, and in the aminoacid sequence of the purine nucleoside phosphorylase of described sudden change, the 97th Asp sports Tyr.
2. purine nucleoside phosphorylase as claimed in claim 1, is characterized in that, the purine nucleoside phosphorylase of described sudden change has following one or more characteristics:
(a), than enzyme work >=30U/mg, be preferably >=45U/mg;
(b) be stored in alkaline environment;
(c) deactivation temperature >=60 ℃;
(d) at 0-55 ℃, shelf time >=3h.
3. the polynucleotide of a separation, is characterized in that, the purine nucleoside phosphorylase of the described sudden change of described polynucleotide encoding claim 1.
4. a carrier, is characterized in that, described carrier contains polynucleotide claimed in claim 3.
5. a host cell, is characterized in that, described host cell comprises in carrier claimed in claim 4 or genome and is integrated with polynucleotide claimed in claim 3.
6. host cell as claimed in claim 5, is characterized in that, described host cell is intestinal bacteria (E.coli) cells.
7. the preparation method of the purine nucleoside phosphorylase of the described sudden change of claim 1, is characterized in that, comprises the following steps:
(a) under conditions suitable for the expression, cultivate host cell claimed in claim 5, thereby give expression to the purine nucleoside phosphorylase of sudden change claimed in claim 1;
(b) separate the purine nucleoside phosphorylase of described sudden change.
8. preparation method as claimed in claim 7, is characterized in that, in step (a) before, described method is further comprising the steps of:
(1) provide the polynucleotide sequence of the purine nucleoside phosphorylase of a Pseudoalteromonas;
(2) by the codon that is coding Tyr corresponding to the codon mutation of the 97th Asp in the aminoacid sequence of the purine nucleoside phosphorylase of wild-type Pseudoalteromonas in described polynucleotide sequence, and optionally, by coded amino acid Arg2 in described polynucleotide sequence, Leu24, Leu77, Ile87, one or more codon AGG in Leu134 and Leu145, CTA, CTA, ATA, CTA replaces with the corresponding preference codon of intestinal bacteria with CTA, thereby make the polynucleotide sequence of the purine nucleoside phosphorylase of coding sudden change claimed in claim 1,
(3) described polynucleotide sequence previous step made imports host cell, thereby makes host cell claimed in claim 5.
9. a method of transforming purine nucleoside phosphorylase, is characterized in that, described method comprises step:
(1) provide the polynucleotide sequence of the purine nucleoside phosphorylase of a Pseudoalteromonas;
(2) by the codon that is coding Tyr corresponding to the codon mutation of the 97th Asp in the aminoacid sequence of the purine nucleoside phosphorylase of wild-type Pseudoalteromonas in described polynucleotide sequence, thereby make the polynucleotide sequence of the purine nucleoside phosphorylase of encoding mutant;
(3) express the described polynucleotide sequence that previous step makes, thereby make the purine nucleoside phosphorylase of sudden change.
10. the purposes of the purine nucleoside phosphorylase of a sudden change claimed in claim 1, is characterized in that, for the catalysis inosine, carries out the phosphorolysis reaction; Or with making the catalyzer that inosine is carried out to the phosphorolysis reaction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310451323.8A CN103468656B (en) | 2013-09-27 | 2013-09-27 | Purine nucleoside phosphorylase and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310451323.8A CN103468656B (en) | 2013-09-27 | 2013-09-27 | Purine nucleoside phosphorylase and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103468656A true CN103468656A (en) | 2013-12-25 |
| CN103468656B CN103468656B (en) | 2015-06-03 |
Family
ID=49793698
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310451323.8A Active CN103468656B (en) | 2013-09-27 | 2013-09-27 | Purine nucleoside phosphorylase and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103468656B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108424943A (en) * | 2017-12-22 | 2018-08-21 | 上海兆维科技发展有限公司 | A method of production 2 '-deoxidations -2 '-fluoro-beta-D-arabinose adenylate |
| CN117070491A (en) * | 2023-10-17 | 2023-11-17 | 吉林凯莱英制药有限公司 | Purine nucleoside phosphorylase mutant and preparation method of 2' -fluoronucleoside |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101629169A (en) * | 2009-08-06 | 2010-01-20 | 杭州利安生物科技有限公司 | Purine nucleoside phosphorylase (PNP) with modified molecules and preparation method thereof |
-
2013
- 2013-09-27 CN CN201310451323.8A patent/CN103468656B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101629169A (en) * | 2009-08-06 | 2010-01-20 | 杭州利安生物科技有限公司 | Purine nucleoside phosphorylase (PNP) with modified molecules and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| JOHANNA D. STOECKLER，ET AL: "Purine Nucleoside Phosphorylase. 3. Reversal of Purine Base Specificity by Site-Directed Mutagenesis", 《BIOCHEMISTRY》, vol. 36, 31 December 1997 (1997-12-31) * |
| MICHELLE R，ET AL: "Molecular Analysis of Mutations in a Patient with Purine Nucleoside Phosphorylase Deficiency", 《AM.J.HUM.GENET》, 31 December 1992 (1992-12-31) * |
| SUWIPA SAEN-OON，ET AL: "Remote Mutations and Active Site Dynamics Correlate with Catalytic Properties of Purine Nucleoside Phosphorylase", 《BIOPHYSICAL JOURNAL》, vol. 94, 31 May 2008 (2008-05-31) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108424943A (en) * | 2017-12-22 | 2018-08-21 | 上海兆维科技发展有限公司 | A method of production 2 '-deoxidations -2 '-fluoro-beta-D-arabinose adenylate |
| CN108424943B (en) * | 2017-12-22 | 2021-06-08 | 上海兆维科技发展有限公司 | Method for producing 2 '-deoxy-2' -fluoro-beta-D-arabinosyladenylate |
| CN117070491A (en) * | 2023-10-17 | 2023-11-17 | 吉林凯莱英制药有限公司 | Purine nucleoside phosphorylase mutant and preparation method of 2' -fluoronucleoside |
| CN117070491B (en) * | 2023-10-17 | 2024-01-19 | 吉林凯莱英制药有限公司 | Purine nucleoside phosphorylase mutant and preparation method of 2' -fluoronucleoside |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103468656B (en) | 2015-06-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TWI704227B (en) | A composition for producing tagatose and methods for producing tagatose using the same | |
| CN105950640B (en) | The kappa-carrageenan enzyme gene and recombinase preparation method obtained using marine bacteria as source | |
| TW201842188A (en) | A composition for producing tagatose and methods for producing tagatose using the same | |
| CN109943546B (en) | Glutamine transaminase mutant and preparation method and application thereof | |
| CN106591271A (en) | Arginine deiminase mutant with improved enzyme activity and temperature stability and application of mutant | |
| CN104531628A (en) | Alcohol dehydrogenase mutant and application thereof | |
| CN106062188B (en) | The L-arabinose isomerase variant of activity of conversion with raising and the method for producing D-Tag using the variant | |
| CN102517271B (en) | Mutant nitrile hydratase | |
| CN101993863B (en) | Glucamylase as well as encoding gene and application thereof | |
| CN101429517A (en) | High temperature resistant xylosidase XynB1, gene encoding the enzyme and uses thereof | |
| CN103468656B (en) | Purine nucleoside phosphorylase and preparation method thereof | |
| CN111621488B (en) | Heat-adaptability-improved inulase exonuclease mutant MutQ23 delta 11 | |
| CN112111472B (en) | Novel beta-xylosidase and preparation thereof | |
| CA3015203C (en) | Novel polyphosphate-dependent glucokinase and method for preparing glucose 6-phosphate by using same | |
| CN101544969B (en) | Mutant of D-carbamyl hydrolysis enzyme and application thereof | |
| CN101215554A (en) | Fire resistant L-arabinose isomerase and gene sequence thereof | |
| CN112921025B (en) | Mutant of epimerase, coding gene, amino acid sequence and application thereof | |
| KR101877120B1 (en) | Process for preparing 2'-Deoxycytidine using bioconversion | |
| CN108823186A (en) | A kind of thermophilic acidic uncooked amylum alpha-amylase mutant and its preparation method and application that cornstarch degradation capability improves | |
| KR20160111947A (en) | Method of producing a recombinant microorganism | |
| Wang et al. | Expression and characterization of an α-glucosidase from Thermoanaerobacter ethanolicus JW200 with potential for industrial application | |
| CN110616211B (en) | Alpha-amylase, coding gene, vector, host and application thereof | |
| CN115747235B (en) | Novel glycoside hydrolase DogH gene and method for improving biological stress resistance thereof | |
| CN105316271A (en) | Method for constructing bacterium strain with high output of rhamnolipid | |
| CN101429518A (en) | High temperature resistant xylosidase XynB2, gene encoding the enzyme and uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 201100 Shanghai East Road, Minhang District, No. 85 Patentee after: SHANGHAI AILEX TECHNOLOGY Co.,Ltd. Address before: 201100 Shanghai East Road, Minhang District, No. 85 Patentee before: SHANGHAI AILEX TECHNOLOGY Co.,Ltd. |
|
| CP03 | Change of name, title or address |
Address after: 201108 Lane 152, 468, Beihengsha River Road, Minhang District, Shanghai Patentee after: AILEX TECHNOLOGY GROUP Co.,Ltd. Address before: No. 85 Youdong Road, Minhang District, Shanghai 201100 Patentee before: SHANGHAI AILEX TECHNOLOGY Co.,Ltd. |
|
| CP03 | Change of name, title or address | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20181212 Address after: 201108 Lane 152, 468, Beihengsha River Road, Minhang District, Shanghai Co-patentee after: ZHEJIANG AILEX MEDICAL Co.,Ltd. Patentee after: AILEX TECHNOLOGY GROUP Co.,Ltd. Address before: 201108 Lane 152, 468, Beihengsha River Road, Minhang District, Shanghai Patentee before: AILEX TECHNOLOGY GROUP Co.,Ltd. |
|
| TR01 | Transfer of patent right |