CN106589104A - Archease and expression and purification, crystal structure as well as application of Archease - Google Patents
Archease and expression and purification, crystal structure as well as application of Archease Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to Archease and expression and purification, a crystal structure as well as application of Archease. The Archease provided by the invention consists of 179 amino acids, and the molecular weight is 20.7kDa; and the Archease can be subjected to soluble expression in an escherichia coli expression strain (i)Bl21(DE3) plus(/i), and has a higher expression quantity. Dynamic light scattering shows that the particle size of Archease becomes large after Archease is in combination with RNA shearing ligase cofactor GTP, and is closely related to function exertion. The resolution ratio of the crystal structure of the Archease is 1.96 angstroms. The structure is formed by 7 beta-pleated sheets and 4 alpha helixes. The Archease plays an important catalytic regulation function in assistance of same operon proteins such as RNA shearing ligase and transmethylase, and is necessary for mature processing of endoplasmic reticulum stress protein XBP1 mRNA. The analysis of the three-dimensional crystal structure of the Archease is beneficial to illuminating the mechanism of action, so that the Archease can be well applied and the action range of Archease can be expanded.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of people source molecular chaperoneses sample protein A rchease and its expression
The important function of purification, crystal structure, dynamic light scattering result and its aspect that plays a role in auxiliary multiple protein.
Background technology
Archease is highly conserved from extinct plants and animal to people, and concrete substrate specificity is still undiscovered, people source Archease energy
Enough it is catalyzed RNA shearing ligase RTCB to play a role, the regeneration that Archease can hinder optic nerve axons is lacked in fruit bat, it is thermophilic
Heat Gu bacterium Archease can be catalyzed transmethylase and play a role.Can be with significantly accelerated RNA in the presence of Archease
Shearing ligase and transmethylase play a role.According to the existing albumen for reporting Archease pair and its same operator
The effect of catalyst has been arrived, and with many albumen of the Archease in same operator for the carrying out of biochemical reaction is with weight
The regulating and controlling effect wanted, in the ripe course of processing of binding protein XBP1mRNA, the assosting effect of Archease is required
's.Archease auxilins have played important function in immunologic process.Therefore obtained by structure biology method
The crystal structure of Archease and show the change of the particle diameter after with reference to cofactor for finding which using dynamic light scattering result
Substrate specificity, illustrates its mechanism of action so as to further widen its application with important directive function.
The content of the invention
It is an object of the invention to raiser sources molecular chaperoneses sample protein A rchease and its expression and purification, crystal structure,
The application of dynamic light scattering result and its aspect that plays a role in auxiliary multiple protein.
The present invention relates to codon optimization is carried out to people source Archease, to obtain in escherichia coli with soluble form
The recombiant protein of expression；Purification, crystallization are carried out to recombiant protein；People source Archease the 20th is obtained to the 179th amino acids
Three-dimensional crystalline structure, and its application.
The present invention has carried out codon optimization to people source Archease first, obtains the people source of codon optimization
Archease.Specifically its full-length gene is cloned on pet28b+ expression vectors, from Nco1 and Xho1 restriction enzyme sites, to keep away
Exempt from the generation of frameshift mutation, the alanine that the addition after start codon ATG is started with guanine, in description process of the present invention
In other amino acid sequences postpone successively.Retain the 6XHis at C- ends on carrier simultaneously, work for subsequent purification.
People source Archease according to the present invention, containing 179 aminoacid, molecular weight is 20.7kDa, and its aminoacid sequence is such as
Shown in SEQ.ID.NO.1.
Prioritizing selection of the present invention using colibacillary procaryotic cell expression system (but be not excluded for other expression systems,
Such as expressed in other antibacterials or other eukaryotic cells)；To the above albumen in the way of fusion protein
Expressed, prioritizing selection His labels have obtained solubility expression and (but have been not excluded for other labels, the label such as such as MBP or GST
Also there is solubility expression).
The method that the present invention also provides expression and Purification of Human source Archease, the method include：Construct people source
Archease recombinant vectors, convert escherichia coli with the carrier, so as to express the recombinant protein A rchease with label, will be upper
((separation principle is for preferred Ni affinity chromatographs by specificity and the method for the special tag recognition to state the albumen of acquisition：It is low
Imidazole concentration buffer carries out loading, and high imidazole concentration buffer carries out eluting)), isolate destination protein, Jing Gel filtrations
Analysis, obtains highly purified recombinant protein A rchease.
Wherein, the level pad prioritizing selection 50mM Tris of affinity chromatograph, pH 8.0；500mM NaCl；5% glycerol；
10mM imidazoles；2mMβ-Me；0.2mM PMSF, elution buffer prioritizing selection 50mM Tris, pH 8.0；500mM NaCl；5%
Glycerol；250mM imidazoles；2mMβ-Me.The buffer prioritizing selection 20mM Tris of gel permeation chromatography, pH7.4；100mM
NaCl；2mM DTT.
Wherein, expression plasmid is pET28b (+), and method for transformation is CaCl2Conversion method, clone strain are escherichia coli
Top10, expression strain are e. coli bl21 (DE3) plus.
The present invention also provides the method crystallized to people source Archease, and methods described includes：By people source Archease
10-30mg/ml being concentrated into, crystal growth condition being screened with vapor phase grafting at 4-30 DEG C, prioritizing selection is Celsius using 15-20
Degree.
The present invention also provides the crystal of the good people source Archease of diffraction property.
The present invention also provides the crystal three dimensional structure of people source Archease, and the structure describes two grades of people source Archease
Structure composition, peptide chain trend, enterprise schema and three-dimensional molecular construction.Wherein diffraction is carried out for people source Archease albumen,
The crystal diffraction data of people source Archease albumen are obtained, to come from hyperthermophilic archaeon strain Archease (PDB ID:It is 4N2P) mould
Type carries out structure elucidation, finally gives the crystal three dimensional structure of people source Archease albumen.
In a particular embodiment, there is provided the crystal three dimensional structure of people source Archease, wherein the crystal is three-dimensional
Atom in structure has a listed at least one of atomic coordinates in table 1, or any with least 40% ammonia in the coordinate
The average root variance of the three-dimensional atomic coordinate of the main chain carbon skeleton of base acid residue is less than or equal toStructure.
The crystal three dimensional structure of the people source Archease that the present invention is obtained, resolution isIts structure space group is P
21 21 21, cell parameter is：α=β=γ=90 °.
In a specific embodiment, the three dimensional structure of described people source Archease is in the asymmetric list of crystallography
There are two protein moleculars in position.The structure is made up of 7 β-pleated sheets and 4 α spirals altogether, wherein, during 7 described β-pleated sheets are located at
The heart, 4 described α are spiraled about around β-pleated sheet.Wherein α spirals 1, i.e., the aminoacid section containing Glu28-Lys36, β foldings
Folded 1, i.e., the aminoacid section containing Tyr44-Trp58, α spirals 2, i.e., the aminoacid section containing Leu62-Met77, α spirals 3, i.e.,
Aminoacid section containing Thr80-Thr82, β-pleated sheet 2, i.e., the aminoacid section containing Gln87-Gln94, α spirals 4 contain
The aminoacid section of Leu98-Ser114, β-pleated sheet 3, i.e., the aminoacid section containing Phe119-Asp130, β-pleated sheet 4 contain
The aminoacid section of Lys135-Glu144, β-pleated sheet 5, i.e., the aminoacid section containing Val156-IIe159, β-pleated sheet 6 contain
The aminoacid section of Gln165-Tyr167, β-pleated sheet 7, i.e., the aminoacid section containing Glu173-IIe180.
Archease is highly conserved from extinct plants and animal to people, according to announcing archeobacteria Archease structure alignments,
Archease the 51st aspartic acid in people source is metal ion binding site, and the 156th lysine is highly conserved, and its metal is tied
The impact to catalytic reaction is detected after closing site and highly conserved site mutation, contributes to illustrating its mechanism of action.
Dynamic light scattering DLS results show, Archease particle diameters after with reference to cofactor GTP become big, complex it is homogeneous
Property is significantly improved compared with the homogeneity of albumen itself.
Research shows that Archease is aiding in same operator albumen such as RNA shearings ligase, transmethylase performance work
Important catalysis adjustment effect is played with aspect, be binding protein XBP1mRNA the ripe course of processing in institute it is required.
The three-dimensional crystalline structure of parsing Archease contributes to illustrating its mechanism of action, so as to preferably playing and widening which
Sphere of action.
Description of the drawings
The three dimensional structure ribbon schematic diagram of Fig. 1 behaviours source Archease.α spirals are represented with blueness；The purplish red color table of β-pleated sheet
Show；Random coil pansy color table shows.Wherein, A is two schematic arrangements in isomerization unit；B is tied for monomer
Structure schematic diagram.
The topological structure schematic diagram of Fig. 2 behaviours source Archease.Wherein, cylindric for α spirals, arrow shaped is β-pleated sheet.Number
Word represented amino acid resi-dues.
The protein purification figure of Fig. 3 behaviours source Archease.Wherein, A：Polyacrylamide Gel Electrophoresis figure；B：Gel
Filtration chromatography analysis chart.Superdex200 16/600, GE Healthcare.
Fig. 4 behaviours source Archease dynamic light scatterings (DLS) testing result.
Fig. 5 behaviours source Archease+GTP (mol ratios 1:5) dynamic light scattering (DLS) testing result.
Specific embodiment
Present disclosure is further illustrated using specific embodiment below.
Herein below is with reference to specific preferred implementation further description made for the present invention, it is impossible to assert
The present invention be embodied as be confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of without departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's
Protection domain.
The expression and purification method of 1. people source Archease of embodiment
People source Archease, shown in its aminoacid sequence SEQ.ID.NO.1；
MKGGSRVSNPAVMAQEEEDVRDYNLTEEQKAIKAKYPPVNRKYEYLDHTADVQLHAWGDTLEEAFEQCA
MAMFGYMTDTGTVEPLQTVEVETQGDDLQSLLFHFLDEWLYKFSADEFFIPREVKVLSIDQRNFKLRSIGWGEEFSL
SKHPQGTEVKAITYSAMQVYNEENPEVFVIIDI
The full-length gene of people source Archease is cloned on the carrier of pET28b (+) by full genome synthesis, carboxyl end
End 6 His of fusion are used for subsequent purification, recombinant vector are transformed in e. coli bl21 (DE3) plus, 37 DEG C of shaken cultivation
To OD600The IPTG of final concentration of 0.5mM is added to carry out abduction delivering during value 0.8 or so, expression condition is：25 DEG C of shaken cultivation
16h。
The recombinant expression carrier for building is transformed in e. coli bl21 (DE3) plus and enters pedestrian source Archease's
Solubility expression.With 50 μ g/ml kanamycin and 34 μ g/ml chloromycetin as selection pressure, switching recombinant strains are in containing
In the LB fluid mediums of above-mentioned selection pressure, 37 DEG C, 220rpm shaken cultivation to OD600Add during value 0.8 or so final concentration of
The IPTG of 0.5mM carries out abduction delivering, 25 DEG C, 200rpm shaken cultivation after 16 to 20 hours 6000rpm centrifugations 15min receive bacterium.
By the expression bacterium being collected by centrifugation with appropriate amount of buffer solution (50mM Tris, pH 8.0；500mM NaCl；5% glycerol；
10mM imidazoles；2mMβ-Me；0.2mM PMSF) outstanding bacterium, using low-temperature ultrahigh-pressure cell crushing instrument, (Guangzhou cumulative biotechnology has
Limit company) cracking thalline, precipitation and other particulate contamination are removed with 18000rpm high speed centrifugations 1h.By centrifuged supernatant with
After Ni-NTA affinity medias are combined, 50mM Tris, pH 8.0 are used；500mM NaCl；5% glycerol；50mM imidazoles；2mM β-Me's
Wash buffer medium, removes foreign protein.50mM Tris, pH 8.0 are used finally；500mM NaCl；5% glycerol；250mM miaows
Azoles；Destination protein is eluted from affinity media by the eluent of 2mM β-Me, and the concentration tube retained with 10KDa is by eluent
Concentration.By the protein solution after concentration, further with gel permeation chromatography, (Superdex200, method purification 16/600) make
Buffer is 20mM Tris, pH7.4；100mM NaCl；2mM DTT.The destination protein for eluting is concentrated into
Concentration is 15-30mg/ml, is preserved in -80 DEG C, for crystallization experiment.
The method for crystallising of 2. people source Archease of embodiment
As above method expression and purification good people source Archease is concentrated into into concentration about 10-30mg/ml, using crystalline reagents
Box (from Crystal Screen Kit I/II, Index, the Wizard Kit 1＆2 of the companies such as Hampton Research,
Wizard Kit 3＆4 etc.) as the primary dcreening operation condition of crystal growth, crystallized using hanging drop air plllutant method.In multiple differences
Crystalline reagents under the conditions of obtain primary crystalline.By optimizing and revising for later stage, preferred 15-20% (w/v) PEG6000,
Used as crystal growth condition, collect a set of resolution is the buffer of 100mM sodium citrate pH5.0-6.0X-ray
Diffraction data.
The receipt of 3. people source Archease of embodiment is collected and structure elucidation
It is after the crystal for preparing is processed as antifreezing agent by the present inventor using 25% glycerol, frozen in liquid nitrogen.Crystal X is penetrated
The data collection of line diffraction is carried out at SSRF (SSRF) biological macromolecule crystal light beam line station (five lines, six station BL18U).
Data processing uses HKL3000, and structure elucidation is coming from hyperthermophilic archaeon strain Archease (PDB ID:It is 4N2P) model, adopts
Molecular replacement technique, using Refmac programs and is aided with Coot softwares and does and further correct, and final parsing obtains people source Archease
The crystal structure of albumen.One of ordinary skill in the art understands that the atom in wherein described crystal three dimensional structure has table 1
In listed at least 40% atomic coordinates, or in the crystal three dimensional structure of 3D albumen at least 40% aminoacid main chain carbon bone
The average root variance of the coordinate in the atomic structure coordinate of frame and table 1 is less than or equal toAtomic coordinates, can be considered as
There is identical structure with 3D albumen.
Specifically, the crystal three dimensional structure of described people source Archease, has two in a crystallography asymmetry unit
Individual protein molecular.It is made up of 7 β-pleated sheets and 4 α spirals, wherein, 7 described β-pleated sheets are located at center, 4 described α spiral shells
Rotation is centered around around β-pleated sheet.Wherein α spirals 1, i.e., the aminoacid section containing Glu28-Lys36, β-pleated sheet 1, i.e., containing Tyr44-
The aminoacid section of Trp58, α spirals 2, i.e., the aminoacid section containing Leu62-Met77, α spirals 3, i.e., containing Thr80-Thr82's
Aminoacid section, β-pleated sheet 2, i.e., the aminoacid section containing Gln87-Gln94, α spirals 4, the i.e. aminoacid containing Leu98-Ser114
Section, β-pleated sheet 3, i.e., the aminoacid section containing Phe119-Asp130, β-pleated sheet 4, i.e., the aminoacid area containing Lys135-Glu144
Section, β-pleated sheet 5, i.e., the aminoacid section containing Val156-IIe159, β-pleated sheet 6, i.e., the aminoacid section containing Gln165-Tyr167,
β-pleated sheet 7, i.e., the aminoacid section containing Glu173-IIe180.
The atomic coordinates group of 1 people source Archease crystal of table is as follows：
Crystal space group：P 21 21 21
Cell parameter is：α=β=γ=90 °.
I/σ：10.23(1.74)
Rwork/Rfree is respectively：0.1906 and 0.2314
Resolving range50-1.96
Data integrity (%)：100.
SEQUENCE LISTING
<110>Fudan University
<120>People source molecular chaperoneses sample albumen and its expression and purification, crystal structure and application
<130> 001
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 179
<212> PRT
<213>
<400> 1
Met Lys Gly Gly Ser Arg Val Ser Asn Pro Ala Val Met Ala Gln Glu
1 5 10 15
Glu Glu Asp Val Arg Asp Tyr Asn Leu Thr Glu Glu Gln Lys Ala Ile
20 25 30
Lys Ala Lys Tyr Pro Pro Val Asn Arg Lys Tyr Glu Tyr Leu Asp His
35 40 45
Thr Ala Asp Val Gln Leu His Ala Trp Gly Asp Thr Leu Glu Glu Ala
50 55 60
Phe Glu Gln Cys Ala Met Ala Met Phe Gly Tyr Met Thr Asp Thr Gly
65 70 75 80
Thr Val Glu Pro Leu Gln Thr Val Glu Val Glu Thr Gln Gly Asp Asp
85 90 95
Leu Gln Ser Leu Leu Phe His Phe Leu Asp Glu Trp Leu Tyr Lys Phe
100 105 110
Ser Ala Asp Glu Phe Phe Ile Pro Arg Glu Val Lys Val Leu Ser Ile
115 120 125
Asp Gln Arg Asn Phe Lys Leu Arg Ser Ile Gly Trp Gly Glu Glu Phe
130 135 140
Ser Leu Ser Lys His Pro Gln Gly Thr Glu Val Lys Ala Ile Thr Tyr
145 150 155 160
Ser Ala Met Gln Val Tyr Asn Glu Glu Asn Pro Glu Val Phe Val Ile
165 170 175
Ile Asp Ile
Claims (7)
1. a kind of people source molecular chaperoneses sample protein A rchease, it is characterised in that the codon of Archease is optimized, i.e.,
Its full-length gene is cloned on pet28b+ expression vectors, from Nco1 and Xho1 restriction enzyme sites, after start codon ATG
Add the alanine started with guanine, other amino acid sequences postpone successively, while retaining the 6XHis at C- ends on carrier；Institute
People source Archease is stated, containing 179 aminoacid, molecular weight is 20.7kDa, and its aminoacid sequence is as shown in SEQ.ID.NO.1.
2. the expression of molecular chaperoneses sample protein A rchease in people source as claimed in claim 1 and purification process, it is characterised in that
Including：People source Archease recombinant vectors are built, and escherichia coli are converted with the carrier, so as to express the recombiant protein with label
Archease, by method with the special tag recognition of the albumen of above-mentioned acquisition by specificity, isolates destination protein,
Jing gel permeation chromatographies, obtain highly purified recombinant protein A rchease；
Wherein, expression plasmid is pET28b (+), and method for transformation is CaCl2Conversion method, clone strain be escherichia coli Top10, table
It is e. coli bl21 (DE3) plus up to bacterial strain.
3. the method for crystallising of molecular chaperoneses sample protein A rchease in people source as claimed in claim 1, it is characterised in that include：
People source Archease is concentrated into into 10-30mg/ml, crystal growth condition is screened with vapor phase grafting under 4-30 °C.
4. the crystal three dimensional structure of molecular chaperoneses sample protein A rchease in people source as claimed in claim 1, it is characterised in that brilliant
Atom in body three dimensional structure has in table 1 listed at least 40% atomic coordinates, or at least main chain carbon of 40% aminoacid
The average root variance of the coordinate in the atomic structure coordinate and table 1 of skeleton is less than or equal to 1.7.
5. the crystal three dimensional structure of molecular chaperoneses sample protein A rchease in people source according to claim 4, it is characterised in that
Resolution is 1.96, and crystal structure space group is P 21 21 21, an asymmetry unit has two molecules；Cell parameter is：a
=46.321, b=55.921, c=133.485, α=β=γ=90 °.
6. the crystal three dimensional structure of molecular chaperoneses sample protein A rchease in people source according to claim 4, it is characterised in that
It is made up of 7 β-pleated sheets and 4 α spirals；7 β-pleated sheets are located at center, and 4 α are spiraled about around β-pleated sheet；Wherein,
α spirals 1, i.e., the aminoacid section containing Glu28-Lys36, β-pleated sheet 1, i.e., the aminoacid section containing Tyr44-Trp58, α spirals
2, i.e., the aminoacid section containing Leu62-Met77, α spirals 3, i.e., the aminoacid section containing Thr80-Thr82, β-pleated sheet 2 contain
The aminoacid section of Gln87-Gln94, α spirals 4, i.e., the aminoacid section containing Leu98-Ser114, β-pleated sheet 3 contain
The aminoacid section of Phe119-Asp130, β-pleated sheet 4, i.e., the aminoacid section containing Lys135-Glu144, β-pleated sheet 5 contain
The aminoacid section of Val156-IIe159, β-pleated sheet 6, i.e., the aminoacid section containing Gln165-Tyr167, β-pleated sheet 7 contain
The aminoacid section of Glu173- IIe180.
7. the crystal three dimensional structure of the people source molecular chaperoneses sample protein A rchease as described in one of claim 4-6, is aiding in
Catalysis adjustment effect in terms of same operator albumen such as RNA shearing ligase, transmethylase.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109837259A (en) * | 2019-02-24 | 2019-06-04 | 复旦大学 | From the thermophilic esterase crystal three-dimensional structure and expression and purification method of marine bacteria |
| CN109913426A (en) * | 2019-02-25 | 2019-06-21 | 复旦大学 | Application of the Thermophilic Bacteria RNA ligase RTCB in low-temperature catalyzed RNA connection and DNA circle |
| CN110256540A (en) * | 2019-03-22 | 2019-09-20 | 复旦大学 | Expression and purification, crystal structure and the application of mycobacterium tuberculosis ribosomal protein S7 |
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| CN109837259B (en) * | 2019-02-24 | 2022-10-11 | 复旦大学 | Thermophilic esterase crystal three-dimensional structure derived from marine bacteria and expression purification method |
| CN109913426A (en) * | 2019-02-25 | 2019-06-21 | 复旦大学 | Application of the Thermophilic Bacteria RNA ligase RTCB in low-temperature catalyzed RNA connection and DNA circle |
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| CN110256540B (en) * | 2019-03-22 | 2022-10-11 | 复旦大学 | Expression and purification of mycobacterium tuberculosis ribosomal protein S7, crystal structure and application |
| CN117603900A (en) * | 2024-01-19 | 2024-02-27 | 清华大学 | Genetically engineered bacterium and construction method and application thereof |
| CN117603900B (en) * | 2024-01-19 | 2024-04-30 | 清华大学 | Genetically engineered bacterium and construction method and application thereof |
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