CN106834324A - A kind of recombinant expression carrier that can promote solubility expression of protein and improve expression quantity - Google Patents
A kind of recombinant expression carrier that can promote solubility expression of protein and improve expression quantity Download PDFInfo
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Abstract
The application belongs to gene engineering technology field, and in particular to a kind of recombinant expression carrier that can promote solubility expression of protein and improve expressing quantity.Recombinant expression carrier pET21b HEMBP（Pyr）It is prokaryotic expression carrier, after double digestion on the basis of pET21b, restructuring is connected with HE MBP（Pyr）TEV sequences；Especially by construction recombination plasmid pUC57 HEMBP（Pyr）The steps such as Nb, digestion, connection, conversion, screening build and obtain.Application of the recombinant expression carrier in protein expression, can be used to express the multiple proteins such as monoclonal antibody, antigen, polyprotein, c di GMP and c GAMP synzyme.The application by building transformation, build new recombinant expression carrier, can preferably improve expressing quantity and improve albumen can solubility, there is preferably practical value and popularization and application meaning in genetic engineering, protein engineering.
Description
Technical field
The application belongs to gene engineering technology field, and in particular to one kind can promote solubility expression of protein and improve albumen
The recombinant expression carrier of expression quantity.
Background technology
With the development of scientific research in the fields such as genomics, protein science and bioinformatics, recombinant DNA technology extensively should
For the application study of various target proteins, such as biochemistry, structure biology, biotechnology application study.As recombinant DNA
The product that technology is arisen at the historic moment, fusion protein is already the new bio molecule that a class has multi-functional characteristic.DNA recombinates skill
Art is introduced into and realizes identification of the target protein in various host biological systems, transformation, expression, separates and purify, application at present compared with
Many host biological systems have：Bacterium (Escherichia coli), yeast, plant, insect and mammal cell line.In these hosts
In biology Escherichia coli so that its reproduction speed is fast, low production cost, protein yield be high etc., and advantage occupies leading position, in large intestine bar
The protein product expressed in bacterium is also widely used in industrial production, vaccine research and development and the analysis of protein structure detection functionality etc..
However, Escherichia coli are due to having own limitations, such as lack the posttranslational modification to eukaryotic protein, external source
Recombinant protein is most of to be expressed with inclusion bodies.Have been reported that display, the GAP-associated protein GAP of the mankind has 75% can be in Escherichia coli
Express, but only 25% is active solubility expression（B ü ssow K, et.al., Structural genomics of
human proteins--target selection and generation of a public catalogue of
Expression clones, Microb Cell Fact., 2005,5：4~21）.Cannot correctly be folded during protein expression,
Cause a large amount of intermediate accumulation sedimentations and form inclusion body, inclusion body is the bottleneck in recombinant protein production process.Current people
Kinds of schemes has been worked out to solve the limitation and defect of Bacillus coli expression foreign protein, such as transformation of engineering bacteria, induction
Optimization (inducing temperature, induction time, inducer concentrations etc.), molecular chaperones coexpression, addition fusion tag of condition etc..
Fusion tag is usually the polypeptide or protein molecule of the stable meltable expression in Escherichia coli, and the label makes
It is pure with can not only promote coupled target protein correct folding raising solubility expression in Escherichia coli to be also convenient for albumen
Change, the destination protein expressed in addition can be additionally used in identification and functional study.Function according to fusion tag can be classified as
Two kinds：A kind of is the soluble flag of promotion recombinant protein solubility expression, such as glutathione S-transferase (GST), maltose
Associated proteins (MBP), thioredoxin A (TrxA), small ubiquitin sample modified protein (SUMO), steroid isomeras (KSI)
And Trp LE etc.；Another kind is easy for the affinity tag of recombinant protein purification, and affinity tag except applying most polies at present
It is histidine-tagged（His6）Outward, also c-myc, HA, FLAG, 1D4, many poly arginines, Streptavidin combination tag, calcium adjust egg
White Binding peptide etc..Affinity tag and soluble flag can be simultaneously typically added in construction of expression vector in order to recombinant protein
Solubility expression after purify.Correct folding and the functional activity of target protein are may interfere with due to fusion tag, so setting
, it is necessary to design a proteolytic cleavage site between fusion tag and destination protein during meter recombinant expression carrier, will pass through
Corresponding proteolytic cleavage obtains target protein after removing fusion tag.
Marmor erodens (Tobacco etch virus, TEV) protease be at present using more protease it
One, protease high specific identification ENLYFQS/G amino acid sequences；There is no non-specific proteolysis to act on target protein；
TEV protease is still active under conditions of low temperature, different pH buffer, high ionic strength.In addition, TEV protease digestion is
Close to the C-terminal of recognition site, keep the N-terminal of target protein complete after digestion, be a kind of extraordinary endo protease.
His6 labels are one of affinity tags most widely used at present.His6 tag molecule amounts are small, without interference with albumen
Function and structure, therefore the extensive use in Crystallographic Study of His6 labels.In addition, His6 labels can also be by immobilization gold
Belong to ion affinity chromatography to purify the target protein of its fusion.Although His6 labels without interference with albumen function and structure and be easy to
Affinity purification, but fusion His6 labels carry out prokaryotic expression albumen have 30 ~ 50% be inclusion body express（Smyth DR,
Et.al., Crystal structures of fusion proteins with large-affinity tags,
Protein Sci., 2003,12 (7):1313-22）.In addition, with the affinity body of His6 labels mark for body radioactivity
Occurs the phenomenon of radioactive build-up apparently higher than the affinity marked without His6 in nucleic molecular imaging application in body liver
Body（Tolmachev V, et.al., HEHEHE-tagged affibody molecule may be purified by
IMAC, is conveniently labeled with [99(m)Tc(CO)3] (+), and shows improved
Biodistribution with reduced hepatic radioactivity accumulation, Bioconjug
Chem. 2010,21 (11):2013-22）.
MBP (maltose binding protein) is maltose-binding protein, is compiled by Escherichia coli malIE genes
The albumen of code, it is one of member of bacterium maltose movement system.MBP albumen hydrophily is strong, can be merged with multiple protein,
Target protein can be helped correctly to fold as a molecular chaperones, be usually used in the solubility expression of target protein.First will within 1988
MBP is connected and the great expression soluble recombinant protein matter in Escherichia coli as fusion tag with foreign gene, is also current
The auxilin of the more commonly used promotion heterologous recombinant protein solubility expression.Recombinant single chain antibody（scFvs）It is that one kind is being faced
Medicative albumen on bed.But inclusion body is easily formed during the albumen is expressed, so as to cause albumen insoluble,
Without activity.When MBP fusion tags are added in the C-terminal of scFvs, it is found that the albumen is high soluble and active,
And the fusion protein is highly stable.In addition, some albumen are insoluble originally, upper MBP labels are combined in these albumen n ends
The solubility of albumen can also be increased.
But in general, because protein structure differs greatly, it is necessary to select suitable label egg during for different albumen
In vain, could preferably improve its expression quantity and solubility expression effect, thus searching of still needing, design new label labelled protein or
Recombinant vector, so that the need for adapting to different protein expressions.
The content of the invention
Most of in Escherichia coli for current recombinant protein is the defect of inclusion body presence, the invention provides one kind
Recombinant expression carrier pET21b-HEMBP（Pyr）, compared with traditional expression vector, can preferably improve expressing quantity and albumen
Can solubility.
Details are as follows for the technical scheme of the application.
A kind of recombinant expression carrier that can promote solubility expression of protein and expression quantity, the recombinant expression carrier is protokaryon table
Up to carrier, pET21b-HEMBP is named as（Pyr）, the recombinant expression carrier carries out NdeI and BamHI on the basis of pET21b
After double digestion, restructuring is connected with HE-MBP（Pyr）- TEV sequences；Wherein HE is histidine-tagged, the MBP of modified form
(Maltotriose-binding protein) is derived from Pyrococcus furiosus high（Pyrococcus furiosus）Albumen mark
Sign, TEV is Tobacco Etch Virus protease cleavage sequence；Its specific building process is：
（1）Construction recombination plasmid pUC57-HEMBP（Pyr）-Nb
Artificial synthesized HEMBP（Pyr）- Nb gene orders, its base sequence is specific as shown in SEQ ID NO.1, with pUC57 plasmids
It is attached, construction recombination plasmid pUC57-HEMBP（Pyr）-Nb；
（2）Digestion, connects, and detailed process is：
By step（1）In recombinant plasmid pUC57-HEMBP（Pyr）NdeI is respectively adopted for-Nb and plasmid pET21b and XhoI enters
Row double digestion
1% agarose gel electrophoresis is carried out to digestion products, and reclaims digestion products；
Digestion products are attached with T4 DNA ligases, 16 DEG C of connections are overnight；
（3）Convert, screen, detailed process is：
By step（2）In connection product pass through chemical method（CaCl2）Convert to Escherichia coli（E.coli）BL21（DE3）
In, then coat LB solid mediums（Ampicillin, 100 μ g/mL）In, it is inverted overnight incubation；
Picking positive single bacterium colony, is inoculated in and contains ampicillin（100 μg/mL）In the LB fluid nutrient mediums of resistance, 37 DEG C,
220 rpm cultivate 6 h, then, plasmid are extracted according to the specification of SanPrep pillar DNA a small amount of extraction agent boxes；
Plasmid to being extracted carries out the identification of NdeI and BamHI double digestions；
To the correct Escherichia coli containing recombinant plasmid of identification, save backup.
The recombinant expression carrier pET21b-HEMBP that can promote solubility expression of protein and expression quantity（Pyr）In albumen
Application in expression, for the prokaryotic expression system of Escherichia coli, can promote the solubility expression of albumen and improve protein expression
Amount, the albumen is specific for example：Monoclonal antibody（Nb、PCV2VHHC3）, antigen（H5HA10、PCV2b（cap）、NLSFMD、
HAFnt、FMDV98）, polyprotein（Cago60）And c-di-GMP and c-GAMP synzyme（CdiGMP499（Caulobacter
vibrioides）、CdiGMP026（Phenylobacterium zucineum）、Prop acid OAS）Deng.
Recombinant expression carrier pET21b-HEMBP provided herein（Pyr）, HE affinity tags, MBP are contained simultaneously
（Pyr）Enzyme site in soluble flag and TEV, compared to conventionally used sequence label in the prior art, its technical advantage is specific
It is embodied in following aspects：
（1）The Host Strains that Escherichia coli are expressed as a kind of foreign recombinant proteins being most widely used at present, it has necessarily
Own limitations, such as lack posttranslational modification to eukaryotic protein, foreign recombinant proteins major part is with inclusion bodies
Expression, thus it is soluble not high after exogenous protein expression；
A kind of new MBP is employed to overcome this defect, in the application（Maltotriose-binding protein,
Pyrococcus furiosus）Soluble flag, different from traditional MBP labels, the MBP in the application（Pyr）Solubility mark
Label come from fierce fireball bacterium（Pyrococcus furiosus）A kind of maltotriose associated proteins（WP_011013078）, its
Original function is to participate in the atriphos combination transhipment of maltotriose in vivo（ABC transport system）,
Belong to the family of bacterium solute binding proteins I；Based on this new MBP（Pyr）Soluble flag, to promote and improve recombinant protein
Can solubility；
（2）His6 labels are the affinitive layer purification labels that are most widely used at present, its have molecular weight it is small, can be in denaturation
Under the conditions of purifying protein, non-immunogenicity, the advantages of do not influence immune analysis, but this label still suffers from certain weak point,
As easily formed inclusion body based on the recombinant protein expressed by this label, being difficult to dissolve；
Recombinant protein after expression is easily set to form the defect of inclusion body to solve His6 labels, the application is remained to ensuring recombinant protein
On the premise of preferable affinity purification, a kind of new HE affinity tags are employed（HEHEHEHEHEHEHE）；HE labels are by even number
The histidine of position is substituted for a hydrophily glutamic acid higher（HEHEHE-tag）, this replacement ensuring HEHEHE-tag
While the affinity body of mark again may be by immobilized metal affinity chromatography purifying protein, recombinant protein is preferably improve
Solubility expression effect, thus His6 labels can be substituted for the affinity purification of fusion protein；
（3）When in the prior art, using prokaryotic expression fusion protein, the prokaryotic expression carrier for being used is typically free of
TEV protease restriction enzyme site, therefore target protein can not be obtained by cutting off fusion tag；By in recombination expression in the present invention
TEV protease restriction enzyme site is integrated in carrier, more can easily realize cutting off purpose of the fusion tag to obtain target protein.
In general, the application can preferably improve albumen table by building transformation, building new recombinant expression carrier
Up to measure and improve albumen can solubility, there is preferably practical value and popularization and application meaning in genetic engineering, protein engineering.
Brief description of the drawings
Fig. 1 is construction of recombinant plasmid schematic diagram, and wherein A is recombinant expression carrier pET21b-HEMBP（Pyr）Structure stream
Journey schematic diagram；
B is recombinant plasmid expression vector pET21b-HEMBP（Pyr）-H5HA10、pET21b-HEMBP（Pyr）-PCV2b、
pET21b-HEMBP（Pyr）-NLSFMD、pET21b-HEMBP（Pyr）The structure schematic flow sheet of-HAFnt；
Fig. 2 is continuous Fig. 1, and wherein C is to contain 10 kinds of genes of interest（Nb、PCV2VHHC3、H5HA10、PCV2b、HAFnt、
FMDV98、Cago60、CdiGMP499（Caulobacter vibrioides）、CdiGMP026（Phenylobacterium
zucineum）、Prop acid OAS）Recombinant plasmid expression vector structure schematic flow sheet；
D is recombinant expression plasmid pET21b-His6-MBP（Pyr）The structure flow chart of-gene（Gene represents genes of interest
PCV2VHHC3, H5HA10, NLSFMD, HAFnt, FMDV98, Cago60, CdiGMP499 or Prop acid OAS）；
Fig. 3 is constructed recombinant expression plasmid pET21b-HEMBP（Pyr）The electrophoretogram of-Nb（Left figure）And digestion qualification result
（Right figure）, wherein：M: DL5 000 DNA Marker；1: pUC57-HEMBP-Nb（NdeI/XhoI, purpose fragment is long by 1 635
bp）；2:PET21b protoplasm grain；3: pET21b-HEMBP-Nb（NdeI/BamHI, the bp of small pieces segment length 1 242）；
Fig. 4 is constructed recombinant plasmid pET21b-HEMBP（Pyr）The electrophoretogram of-gene and pET21b-His6MBP-gene（A）
And digestion qualification result（B1、B2）；
In A figures：M: DL5 000 DNA Marker；1:PET21b-His6MBP carriers；2: pET21b-HEMBP(Pyr)-Nb
（387 bp）；3:Containing FMDV98（639 bp）The plasmid of gene；4:Containing PCV2b（705 bp）The plasmid of gene；5:Contain
CdiGMP499（714 bp）The plasmid of gene；6:Containing CdiGMP026（648 bp）The plasmid of gene；7:Acid containing Prop
OAS（1 035 bp）The plasmid of gene；8:Containing NLSFMD（672 bp）The plasmid of gene；9:Containing HAFnt（951 bp）Gene
Plasmid；10:Containing H5HA10（522 bp）The plasmid of gene；11:Containing PCV2VHHC3（414 bp）The plasmid of gene；12:
Containing Cago60（801 bp）：The plasmid of gene；
B1 figures are pET21b-HEMBP（Pyr）- gene recombinant plasmid digestion qualification results（NdeI/XhoI）, wherein M: DL5
000 DNA Marker；1: pET21b-HEMBP（Pyr）-Nb（A length of 1 629 bp of small fragment）；2: pET21b-HEMBP
（Pyr）-FMDV98（A length of 1 881 bp of small fragment）；3: pET21b-HEMBP（Pyr）-PCV2b（Small fragment a length of 1 947
bp）；4: pET21b-HEMBP（Pyr）-CdiGMP026（A length of 1 890 bp of small fragment）；5: pET21b-HEMBP（Pyr）-
H5HA10（A length of 1 764 bp of small fragment）；6: pET21b-HEMBP（Pyr）-NLSFMD（A length of 1 914 bp of small fragment）；7:
pET21b-HEMBP（Pyr）-Cago60（A length of 2 043 bp of small fragment）；8: pET21b-HEMBP（Pyr）-Prop acid
OAS（A length of 2 277 bp of small fragment）；9: pET21b-HEMBP（Pyr）-CdiGMP499（A length of 1 956 bp of small fragment）；
10: pET21b-HEMBP（Pyr）-PCV2VHHC3（A length of 1 656 bp of small fragment）；11: pET21b-HEMBP（Pyr）-
HAFnt（A length of 2 193 bp of small fragment）；
B2 figures are pET21b-His6MBP-gene recombinant plasmid digestion qualification results（NdeI/XhoI）, wherein M: DL5 000
DNA Marker；1: pET21b-His6MBP-CdiGMP026（A length of 1 849 bp of small fragment）；2: pET21b-His6MBP-
Cago60（A length of 2 002 bp of small fragment）；3: pET21b-His6MBP-Nb（A length of 1 588 bp of small fragment）；4:
pET21b-His6MBP-FMDV98（A length of 1 840 bp of small fragment）；5: pET21b-His6MBP-PCV2b（Small fragment a length of 1
906 bp）；6: pET21b-His6MBP-CdiGMP499（A length of 1 915 bp of small fragment）；7: pET21b-His6MBP-
Prop acid OAS（A length of 2 236 bp of small fragment）；8: pET21b-His6MBP- NLSFMD（Small fragment a length of 1 873
bp）；9: pET21b-His6MBP-HAFnt（A length of 2 152 bp of small fragment）；10: pET21b-His6MBP-H5HA10（It is small
A length of 1 723 bp of fragment）；11: pET21b-His6MBP-PCV2VHHC3（A length of 1 615 bp of small fragment）；
Fig. 5 is constructed pET21b-His6-MBP（Pyr）- gene recombinant plasmid electrophoretograms（A）And digestion（NdeI/XhoI）Mirror
Determine result（B）；In A figures, M: DL 5000 DNA Marker；1: pET21b-HEMBP（Pyr）-Cago60（Purpose fragment is long by 1
980 bp）；2:pET21b-HEMBP(Pyr)-PCV2VHHC3（A length of 1 593 bp of purpose fragment）；3: pET21b-HEMBP
(Pyr)-FMDV98（Purpose fragment 1 818 bp long）；4: pET21b-HEMBP(Pyr)-H5HA10（Purpose fragment is long by 1 701
bp）；5: pET21b-HEMBP(Pyr)-NLSFMD（Purpose fragment 1 851 bp long） 6: pET21b-HEMBP(Pyr)-
HAFnt（Purpose fragment 2 130 bp long）；7: pET21b-HEMBP(Pyr)-Prop acid OAS（Purpose fragment is long by 2 214
bp）；8: pET21b-HEMBP（Pyr）-CdiGMP499（Purpose fragment 1 893 bp long）；9: pET21b-His6；
In B figures, M: DL5 000 DNA Marker；1: pET21b-His6-MBP(Pyr)-FMDV98（Small fragment a length of 1
839 bp）；2: pET21b-His6-MBP(Pyr)-CdiGMP499（A length of 1 914 bp of small fragment）；3: pET21b-His6-
MBP(Pyr)-PCV2VHHC3（A length of 1 614 bp of small fragment）；4: pET21b-His6-MBP(Pyr)-Prop acid OAS
（A length of 2 235 bp of small fragment）；5: pET21b-His6-MBP(Pyr)-H5HA10（A length of 1 722 bp of small fragment）；6:
pET21b-His6-MBP(Pyr)-HAFnt（A length of 2 151 bp of small fragment）；7: pET21b-His6-MBP(Pyr)-NLSFMD
（A length of 1 872 bp of small fragment）；8: pET21b-His6-MBP(Pyr)-Cago60（A length of 2 001 bp of small fragment）；
Fig. 6 is pET21b-HEMBP（Pyr）Recombinant expression carrier and other expression vectors to the difference of Partial Protein expression of results,
Wherein M: Thermo Scientific PageRuler Prestained Protein Ladder（170 kDa、130 kDa、
100 kDa、70 kDa、55 kDa、40 kDa、35 kDa、25 kDa、15 kDa、10 kDa）；1:Full bacterium is not induced；2:Lure
Full bacterium after leading；3:Supernatant after induction；4:Precipitated after induction；
Fig. 7 is pET21b-HEMBP（Pyr）Recombinant expression carrier（It is left）With pET21b-His6MBP expression vectors（It is right）To part
Protein expression Comparative result, wherein M: Thermo Scientific PageRuler Prestained Protein Ladder
（170 kDa、130 kDa、100 kDa、70 kDa、55 kDa、40 kDa、35 kDa、25 kDa、15 kDa、10 kDa）；1:
Full bacterium is not induced；2:Full bacterium after induction；3:Supernatant after induction；4:Precipitated after induction；
Fig. 8 is pET21b-HEMBP（Pyr）Recombinant expression carrier（It is left）With pET21b-His6MBP（Pyr）Expression vector（It is right）It is right
The expression of results contrast of Partial Protein, wherein M: Thermo Scientific PageRuler Prestained Protein
Ladder（170 kDa、130 kDa、100 kDa、70 kDa、55 kDa、40 kDa、35 kDa、25 kDa、15 kDa、10
kDa）；1:Full bacterium is not induced；2:Full bacterium after induction；3:Supernatant after induction；4:Precipitated after induction；
Fig. 9 is pET21b-His6-MBP（Pyr）Recombinant expression carrier（It is left）With pET21b-His6MBP recombinant expression carriers（It is right）
Contrast to Partial Protein expression of results, wherein M: Thermo Scientific PageRuler Prestained
Protein Ladder（170 kDa、130 kDa、100 kDa、70 kDa、55 kDa、40 kDa、35 kDa、25 kDa、15
kDa、10 kDa）；1:Full bacterium is not induced；2:Full bacterium after induction；3:Supernatant after induction；4:Precipitated after induction.
Specific embodiment
Explanation is described further to the application with reference to embodiment, before specific embodiment is introduced, to following realities
Applying be related to partial material situation to be briefly discussed below in example.
Biomaterial：
PUC57 plasmids, pET21b plasmids, purchased from Novagen companies；
The gene orders such as His6, His6MBP, H5HA10, LSL150-cherry-PCV2b/NLSFMD/HAFnt are by Jin Siruisheng
The synthesis of thing technology company is provided, and then builds pET21b-His6MBP, pET21b-His6, pET21b- according to prior art
The recombinant plasmids such as His6-cherry-H5HA10, pET21b-LSL150-cherry-PCV2b/NLSFMD/HAFnt；
Related gene examining order is also completed by Jin Sirui biotech companies in following embodiments；
Also need to explain, cherry albumen is a kind of red fluorescent protein, can after fusion protein is expressed through inducing soluble
Thalline is presented red in fluorescence, be easy to observation；
Experiment reagent：
BamHI（FD0054）、NdeI（FD0583）、NheI（FD0973）、XhoI（FD0694）, pre-dyed albumen Marker
（PageRuler Prestained Protein Ladder）Deng being Thermo ScientificTM products；
DNA a small amount of extraction agent box, DNA glue reclaim kits, raw work bioengineering（Shanghai）Co., Ltd's product；
T4 DNA ligases（#M0202S）, New England Biolabs Products；
Ampicillin（Amp）, Beijing Suo Laibao Science and Technology Ltd；
DNA molecular quality standard DL5 000, purchased from precious bioengineering (Dalian) Co., Ltd；
Lysozyme, Actibity>20000 u/mg, Beijing Suo Laibao Science and Technology Ltd；
30% acrylamide (29:1), 2 × SDS PAGE electrophoresis sample-loading buffer, isopropyl-D- thiogalactosides
（IPTG）, Beijing DingGuo ChangSheng Biology Technology Co., Ltd's product；
Agarose（Agarose）, purchased from Xi'an bio tech ltd of Guoan；
LB culture mediums：Tryptone（Tryptone） 10 g、Yeast Extract （Yeast extract）5 10 g of g, NaCl,
It is dissolved in 800 mL ultra-pure waters, adjusts pH to 7.0 with NaOH, add water and be settled to 1 L, autoclaving is standby；In solid medium, separately
Plus 12 g agar powders；
PBS（pH=7.4）：NaCl 8 g、KCl 0.2 g、Na2HPO4 1.42 g、KH2The g of PO4 0.27, adjust after being dissolved in water
PH is settled to 1L to 7.4, and room temperature is saved backup after autoclaving；
50 × TAE electrophoretic buffers：Weigh 242g Tris, 37.2g Na2EDTA·2H2O, adds ultra-pure water fully molten
Solution, adds the acetic acid of 57.1 mL fully to mix, and solution is settled into 1 L with ultra-pure water, and room temperature is standby；Matched somebody with somebody with ultra-pure water
It is set to 1 × TAE electrophoretic buffers.
10% ammonium persulfate（APS）：1 g APS are weighed, 10 mL are fully dissolved and be settled to ultra-pure water, 4 DEG C of preservations are standby
With；
5 × SDS-PAGE electrophoretic buffers：Weigh 15.15 g Tris, 5 g neopelexes（SDS）, 72.05
G glycine, adds ultra-pure water fully to dissolve, and is settled to 1 L, and normal temperature is saved backup；
Coomassie brilliant blue R_250 dyeing liquor：The g of coomassie brilliant blue R_250 1 is weighed, the isopropanol of 250 mL is measured, added
100 mL glacial acetic acid, add the ultra-pure water of 650 mL to be sufficiently stirred for dissolving, and removal particulate matter is filtered with filter paper, and room temperature preservation is standby
With；
Coomassie brilliant blue destainer：The mL of absolute ethyl alcohol 100, the mL of glacial acetic acid 100, ultra-pure water are settled to 500 mL, room temperature preservation
It is standby；
Laboratory apparatus：
Electrophoresis apparatus and electrophoresis tank (JY600C), Beijing Jun Yi east electrophoresis equipment Co., Ltd；
The gel imaging systems of Amersham Imager 600, GE Healthcare Life Sciences products；
Ultrasonic cell disruptor, Ningbo Xin Zhike devices research institute；
BioSpectrometer ultraviolet/visible light spectrophotometers, Eppendorf；
Embodiment 1
Solubility expression of protein can be promoted and the recombinant expression carrier pET21b-HEMBP of expressing quantity is improved（Pyr）, build
Flow such as Fig. 1（A）It is shown, it is built-up especially by following steps.
（1）Construction recombination plasmid pUC57-HEMBP（Pyr）-Nb
Artificial synthesized HEMBP（Pyr）- Nb gene orders, particular sequence is connected as shown in SEQ ID NO.1 with pUC57 plasmids
Connect, construction recombination plasmid pUC57-HEMBP（Pyr）-Nb.
（2）Digestion, connects, and detailed process is：
By plasmid pUC57-HEMBP（Pyr）- Nb and plasmid pET21b is respectively adopted NdeI and XhoI carries out double digestion, 30 μ L enzymes
Cut System Design as follows：
NdeI, 1 μ L；
XhoI, 1 μ L；
10 × FastDigest Green Buffer, 3 μ L；
Plasmid pUC57-HEMBP（Pyr）-Nb（Or plasmid pET21b）, 1 μ g；
ddH2O adds to 30 μ L；
37 DEG C of h of digestion 1；
1% agarose gel electrophoresis is carried out to digestion products, and reclaims digestion products；
Digestion products are attached with T4 DNA ligases, 16 DEG C of connections are overnight.
（2）Convert, screen, detailed process is：
By step（1）In connection product pass through chemical method（CaCl2）Convert to Escherichia coli（E.coli）BL21（DE3）
In, then coat LB solid mediums（Amp、100μg/mL）In, it is inverted overnight incubation；
Picking positive single bacterium colony, is inoculated in and contains ampicillin（100 μg/mL）Resistance LB fluid nutrient mediums in, 37 DEG C,
220 rpm cultivate 6 h, then, plasmid are extracted according to the specification of SanPrep pillar DNA a small amount of extraction agent boxes.
Plasmid to being extracted carries out double digestion identification, and 10 μ L digestion System Designs are as follows：
NdeI, 0.25 μ L；
BamHI, 0.25 μ L；
10 × FastDigest Green Buffer, 1 μ L；
Extracted plasmid, 250 ng；
ddH2O adds to 10 μ L；
37 DEG C of h of digestion 1；Digestion products are carried out with 1% agarose gel electrophoresis and separates identification.
Qualification result is as shown in Figure 3.Fig. 3 results show pET21b-HEMBP（Pyr）- Nb is successfully constructed.It is correct to identifying
The Escherichia coli containing recombinant plasmid, save backup.
Embodiment 2
It is the recombinant expression carrier pET21b-HEMBP constructed by further detection judges on the basis of embodiment 1（Pyr）To egg
White expression quantity and albumen can solubility promotion lifting effect, using prokaryotic expression system, inventor enters to different foreign proteins
Expression of having gone is tested, and related experiment process is briefly discussed below.
（One）Build the recombinant expression carrier containing genes of interest
Genes of interest specifically has H5HA10, PCV2b, NLSFMD, HAFnt, with reference to Fig. 1（B）Structure schematic flow sheet, contain mesh
Gene recombinant plasmid expression vector pET21b-HEMBP（Pyr）-H5HA10、pET21b-HEMBP（Pyr）-PCV2b、
pET21b-HEMBP（Pyr）-NLSFMD、pET21b-HEMBP（Pyr）- HAFnt, specific building process is as follows：
With BamHI and XhoI enzymes respectively to the recombinant expression carrier pET21b-HEMBP prepared by embodiment 1（Pyr）- Nb and
pET21b-His6-cherry-H5HA10、pET21b-LSL150-cherry-PCV2b、pET21b-LSL150-cherry-
NLSFMD, pET21b-LSL150-cherry-HAFnt plasmid carry out BamHI and XhoI double digestions（Digestion system reference implementation example
30 μ L digestion systems are designed in 1）；
Digestion products are entered with row agarose gel electrophoresis, and reclaims digestion products；
Digestion products are attached with T4 DNA ligases, and then construction recombination plasmid expression vector pET21b-HEMBP
（Pyr）-H5HA10、pET21b-HEMBP（Pyr）-PCV2b、pET21b-HEMBP（Pyr）-NLSFMD、pET21b-HEMBP
（Pyr）-HAFnt；
Connection product is converted to e. coli bl21（DE3）In, and screened（Screening process reference implementation example 1）, to sun
Property grain carries out double digestion identification using NdeI and XhoI, and digestion qualification result is as shown in Figure 4.
The display construction of recombinant plasmid success of Fig. 4 results.
The correct bacterial strain containing recombinant plasmid of digestion identification is saved backup.
By the constructed bacterial strain containing correct recombinant plasmid and it is original containing pET21b-His6-cherry-H5HA10,
pET21b-LSL150-cherry-PCV2b、pET21b-LSL150-cherry- NLSFMD、pET21b-LSL150-cherry-
The bacterial strain of HAFnt plasmids, carries out induced expression respectively, and thalline is evaluated expressing quantity after taking induction.Detailed process
For：
（1）The activation mL of bacterium solution 1 is taken, by 1:100 volume ratio, is inoculated in the LB fluid nutrient mediums containing the μ g/mL of Amp 100,
37 DEG C, 220 rpm shaken cultivations to OD600=0.7 or so；
（2）0.5 mM IPTG are added, 25 DEG C of induced expressions are overnight.
Prepare respectively and do not induce sample after full bacterium sample and induced expression, it is specific as follows：
1st, full bacterium sample is not induced
Before IPTG induced expressions, bacteria liquid sample is carried out as follows as full bacterium sample is not induced before taking 1 mL induced expressions
Treatment：
The rpm of bacterium solution 9000 is centrifuged 1 min, after abandoning supernatant, plus（OD600Value × 100）1 × loading buffer weights of μ L
Outstanding thalline；99 DEG C are boiled 10min, then 12000 g are centrifuged 2 min, and -20 DEG C preserve standby inspection.
2nd, full bacterium sample after induced expression
The OD of bacterium solution after induced expression is determined with ultraviolet specrophotometer600Value, full bacterium sample preparation is not induced then referring to above-mentioned
Mode prepares full bacterium sample after induced expression.
3rd, deposit sample after Supernatant samples and induced expression after induced expression
Bacterium solution centrifugation after 10 mL inductions is taken, is used（OD600 value × 100/2）The PBS of μ L（pH=7.4）Resuspended thalline, plus lysozyme
After (1 mol/L) ice bath 30 min, low-temperature ultrasonic crushes thalline, and (s of ultrasonic time 4, interval 7 is s)；
It is crushed to 4 DEG C of 12000 g of clarification and 30 min is centrifuged；
Draw the supernatant of 30 μ L and add 2 × loading buffer of equivalent, prepare Supernatant samples after induction；
After abandoning remaining supernatant, plus（OD600 value × 100/2）The resuspended precipitations of μ L PBS, draw the re-suspension liquid of 30 μ L and add equivalent
2 × loading buffer, prepare deposit sample after induction.
Specific method is to be detected to protein content in above-mentioned sample：
12% PAGE gel is prepared, 10 μ L are taken respectively does not induce after full bacterium, induction that supernatant is induced after full bacterium, induction
Deposit sample loading, 160 V are adjusted to when 120 V electrophoresis to sample are into separation gel interface by voltage afterwards, treat dyestuff apart from gel
During 0.5 cm of bottom, electrophoresis terminates；
Concentration glue is peeled off with offset plate is unloaded, the separation gel that will be unloaded is contaminated carries out coomassie brilliant blue staining in dyeing liquor, and room temperature is shaken
2 h of dynamic dyeing；
Gel is immersed into destainer after reclaiming dyeing liquor, destainer is changed clear to the band in glue for several times；
Taken pictures with the gel imaging systems of Amersham Imager 600 and preserve and carry out gray analysis.
Induced expression result is as shown in Figure 6.Analysis shows：pET21b-HEMBP（Pyr）Recombinant expression carrier can make not
HAFnt, PCV2b and H5HA10 solubility expression of protein of NLSFMD albumen and the inclusion body expression of expression.Gray analysis result
Display pET21b-HEMBP（Pyr）- HAFnt/NLSFMD/PCV2b/H5HA10 recombinant proteins can solubility be respectively：75%、
60.7%th, 78% and 82.7%；Wherein can recombinant protein amount in solubility=supernatant/(recombinant protein in recombinant protein amount+precipitation in supernatant
Amount).
Embodiment 3
Based on the recombinant expression carrier constructed by embodiment 1, further, inventor is to 10 kinds of different genes of interest difference
Construct recombinant expression carrier and carried out prokaryotic expression, 10 kinds of genes of interest specifically have：Nb、PCV2VHHC3、H5HA10、
PCV2b、HAFnt、FMDV98、Cago60、CdiGMP499（Caulobacter vibrioides）、CdiGMP026
（Phenylobacterium zucineum）, Prop acid OAS, associative operation reference implementation example 1,2 and prior art are
Can, repeat no more.
Structure flow chart such as Fig. 2 of related recombinant expression plasmid（C）It is shown, the digestion to constructed recombinant expression plasmid
Qualification result is as shown in Figure 4.Result shows that construction of recombinant plasmid is successful.
To further demonstrate that technique effect of the invention, while based on existing pET21b-His6MBP plasmids, ginseng
Embodiment 1,2 associative operations and prior art are examined, above-mentioned 9 kinds of genes of interest are integrated and recombinantly expressed, and in this, as
Comparative example.
To different plasmid expression vectors do not induce full bacterium, induction after deposit sample after supernatant induction after full bacterium, induction
12% SDS-PAGE electrophoresis is carried out, coomassie brilliant blue staining is then carried out.The different recombinant plasmid expression vectors of different genes
Electrophoresis result is as shown in fig. 7, the gray analysis result after gel imaging is as shown in table 1 below.
1 two kinds of expression vectors of table promote solubility expression of protein comparison in difference：
Expression quantity (%) accounts for the percentage of total protein in Escherichia coli for recombinant protein；
Can solubility (%) be that expression quantity in recombinant protein supernatant accounts for the percentage of (expression quantity in expression quantity+precipitation in supernatant)
Than.
With reference to Fig. 7 and the statistics of above-mentioned table 1, further analysis can be seen that：
HEMBP（Pyr）- Nb recombinant proteins can solubility compared with His6MBP-Nb, improve 49.2%；
HEMBP（Pyr）- PCV2b recombinant proteins can solubility compared with His6MBP-PCV2b, improve 11.5%；
HEMBP（Pyr）- Cago60 recombinant proteins can solubility compared with His6MBP-Cago60, improve 28.1%；
HEMBP（Pyr）- PCV2VHHC3 recombinant proteins can solubility compared with pET21b-His6MBP- PCV2VHHC3, improve
17.4%；
HEMBP（Pyr）- CdiGMP499 recombinant proteins can solubility compared with His6MBP- CdiGMP499, improve 25.9%；
HEMBP（Pyr）- Prop acid OAS recombinant proteins can solubility compared with His6MBP- Prop acid OAS, improve
42.7%；
HEMBP（Pyr）- FMDV98 recombinant proteins can solubility compared with His6MBP- FMDV98, improve 42.2%；
HEMBP（Pyr）- H5HA10 recombinant proteins can solubility compared with His6MBP- H5HA10, improve 22.4%；
Without expression, HEMBP after the induction of His6MBP-CdiGMP026 recombinant proteins（Pyr）- CdiGMP026 recombinant proteins have expression
And 35% be solubility expression.
Integrated embodiment 2 and above-mentioned statistics, it can be seen that：For different types of gene, using new pET21b-
HEMBP（Pyr）After recombinant expression carrier, compared to original expression vector, its albumen can solubility and expressing quantity have compared with
It is good to improve, and lifting effect is obvious.
Embodiment 4
On the basis of above-described embodiment, to further demonstrate that the sequence label provided in the present invention entrained by recombinant expression carrier
Technique effect, inventor based on pET21b-His6 vector plasmids, with reference to Fig. 2（D）Recombinant plasmid expression vector structure
Build flow chart, with NheI and XhoI by embodiment 3 with pET21b-HEMBP（Pyr）Based on, it is integrated with genes of interest
（PCV2VHHC3、H5HA10、NLSFMD、HAFnt、FMDV98、Cago60、CdiGMP499、Prop acid OAS）Restructuring matter
Grain carries out double digestion with NheI and XhoI, while carrying out the double digestion of NheI and XhoI to pET21b-His6 vector plasmids；By enzyme
Product is cut to be attached, and convert, screen, digestion identification, build and obtain recombinant expression plasmid pET21b-His6-MBP（Pyr）-
gene（Gene represent genes of interest PCV2VHHC3, H5HA10, NLSFMD, HAFnt, FMDV98, Cago60, CdiGMP499 or
Prop acid OAS）.
Digestion qualification result to recombinant plasmid is as shown in Figure 5.Result display construction of recombinant plasmid success.
Associative operation reference implementation example 1,2,3 and prior art, repeat no more.
Induced expression is carried out to constructed recombinant plasmid expression vector, is taken respectively and is not induced full bacterium after full bacterium, induction, lures
Deposit sample carries out 12% SDS-PAGE electrophoresis after leading rear supernatant induction, and carries out coomassie brilliant blue staining, to protein expression
Amount is measured and analyzes.
Wherein, part genes of interest is using HE-MBP（Pyr）Label and His6-MBP（Pyr）During label, protein expression
The difference results of amount and solubility expression are as shown in Figure 8.As can be seen from the figure：
HE-MBP（Pyr）The expression quantity of-PCV2VHHC3/CdiGMP499/H5HA10 recombinant proteins is significantly higher than His6-MBP
（Pyr）The expression quantity of-PCV2VHHC3/CdiGMP499/H5HA10 recombinant proteins, can solubility be higher than the latter；
HE-MBP（Pyr）The expression quantity of-Cago60/FMDV98/NLSFMD/Prop acid OAS recombinant proteins is slightly above His6-
MBP（Pyr）The expression quantity of-Cago60/FMDV98/NLSFMD/Prop acid OAS recombinant proteins, can solubility be respectively increased
8.9%th, 19.5%, 4.8% and 15.2%.
Part genes of interest uses His6-MBP（Pyr）Label and His6MBP labels are to recombinant protein expression quantity and solvable
Property expression influence difference it is as shown in Figure 9.Fig. 9 results show：
His6-MBP-HAFnt expression of recombinant proteins amount is higher than HE-MBP（Pyr）- HAFnt expression of recombinant proteins amounts, but only
59.4% can solubility, and HE-MBP（Pyr）- HAFnt recombinant proteins whole solubility expression；
His6-MBP-Prop acid OAS/H5HA10 expression of recombinant proteins amounts are also above HE-MBP（Pyr）-Prop acid
OAS/H5HA10 expression of recombinant proteins amounts, but can solubility be respectively 32% and 57.1%, less than HE-MBP（Pyr）-Prop acid
OAS/H5HA10 recombinant proteins can solubility, respectively 59.5% and 71.7%；
HE-MBP（Pyr）- FMDV98/Cago60 expression of recombinant proteins amount compares without bright with His6-MBP- FMDV98/Cago60
Significant difference is different, but can solubility be above the latter.
To the gray analysis result table 2 specific as follows after protein expression, shown in table 3.
Table 2： pET21b-HE-MBP（Pyr）With pET21b-His6-MBP（Pyr）Expression vector promotes albumen solubility table
Up to comparison in difference：
Expression quantity (%) accounts for the percentage of total protein in Escherichia coli for recombinant protein；
Can solubility (%) be that expression quantity in recombinant protein supernatant accounts for the percentage of (expression quantity in expression quantity+precipitation in supernatant)
Than.
Table 3： pET21b-His6-MBP（Pyr）Promote solubility expression of protein poor with pET21b-His6MBP expression vectors
Different comparing：
Expression quantity (%) accounts for the percentage of total protein in Escherichia coli for recombinant protein；
Can solubility (%) be that expression quantity in recombinant protein supernatant accounts for the percentage of (expression quantity in expression quantity+precipitation in supernatant)
Than.
SEQUENCE LISTING
<110>Agricultural University Of He'nan
<120>A kind of recombinant expression carrier that can promote solubility expression of protein and improve expression quantity
<130> none
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1242
<212> DNA
<213>Engineer
<400> 1
catatgaaat attataaaca cgagcacgag catgagcacg aacacgagca cgaacacgag 60
ggtgctagcg gcatgaagat cgaggaaggt aaggttgtta tttggcacgc gatgcagccg 120
aacgaactgg aagtgtttca aagcctggcg gaggaataca tggcgctgtg cccggaagtg 180
gagatcgttt tcgagcagaa gccgaacctg gaagacgcgc tgaaagcggc gattccgacc 240
ggtcagggtc cggacctgtt tatctgggcg cacgattgga ttggtaaatt cgcggaggcg 300
ggtctgctgg aaccgatcga cgagtacgtt accgaagatc tgctgaacga atttgcgccg 360
atggcgcagg atgcgatgca atacaagggt cactactatg cgctgccgtt cgcggcggag 420
accgtggcga tcatttataa caaggagatg gttagcgaac cgccgaaaac ctttgacgag 480
atgaaggcga ttatggaaaa atactatgat ccggcgaacg aaaaatacgg catcgcgtgg 540
ccgattaacg cgtatttcat cagcgcgatt gcgcaggcgt ttggtggcta ctatttcgac 600
gataagaccg agcaaccggg tctggacaaa ccggaaacca tcgagggctt taagttcttt 660
ttcaccgaaa tttggccgta catggcgccg accggtgatt ataacaccca gcaaagcatc 720
ttcctggagg gtcgtgcgcc gatgatggtg aacggcccgt ggagcatcaa cgacgttaag 780
aaagcgggta ttaacttcgg cgtggttccg ctgccgccga tcattaagga tggtaaagaa 840
tactggccgc gtccgtatgg tggcgtgaaa ctgatctact ttgcggcggg cattaagaac 900
aaagacgcgg cgtggaagtt cgcgaaatgg ctgaccacca gcgaggaaag catcaagacc 960
ctggcgctgg agctgggtta tattccggtg ctgaccaaag ttctggacga tccggaaatc 1020
aagaacgacc cggtgattta cggttttggc caggcggttc aacacgcgta tctgatgccg 1080
aagagcccga aaatgagcgc ggtgtggggt ggcgttgatg gtgcgatcaa cgagattctg 1140
caggacccgc aaaacgcgga tatcgagggc attctgaaga aataccagca agaaatcctg 1200
aacaacatgc aggaggaaaa cctgtacttc caatccggat cc 1242
Claims (4)
1. it is a kind of can promote solubility expression of protein and improve expression quantity recombinant expression carrier, it is characterised in that the restructuring table
Up to carrier pET21b-HEMBP（Pyr）It is prokaryotic expression carrier, builds obtain as follows：
（1）Construction recombination plasmid pUC57-HEMBP（Pyr）-Nb
Artificial synthesized HEMBP（Pyr）- Nb gene orders, its base sequence is specific as shown in SEQ ID NO.1, with pUC57 plasmids
It is attached, construction recombination plasmid pUC57-HEMBP（Pyr）-Nb；
（2）Digestion, connects, and detailed process is：
By step（1）In recombinant plasmid pUC57-HEMBP（Pyr）NdeI is respectively adopted for-Nb and plasmid pET21b and XhoI enters
Row double digestion；Reclaim digestion products；Digestion products are attached；
（3）Convert, screen, detailed process is：
By step（2）In connection product convert to e. coli bl21 competent cell, carry out resistance screening；Picking is positive single
Bacterium colony, the identification of NdeI and BamHI double digestions is carried out after extracting plasmid；Identification is correctly recombinant expression carrier pET21b-
HEMBP（Pyr）.
2. solubility expression of protein can be promoted described in claim 1 and the recombinant expression carrier pET21b-HEMBP of expression quantity is improved
（Pyr）Construction method, it is characterised in that specifically include following steps：
（1）Construction recombination plasmid pUC57-HEMBP（Pyr）-Nb
Artificial synthesized HEMBP（Pyr）- Nb gene orders, its base sequence is specific as shown in SEQ ID NO.1, with pUC57 plasmids
It is attached, construction recombination plasmid pUC57-HEMBP（Pyr）-Nb；
（2）Digestion, connects, and detailed process is：
By step（1）In recombinant plasmid pUC57-HEMBP（Pyr）NdeI is respectively adopted for-Nb and plasmid pET21b and XhoI enters
Row double digestion；Reclaim digestion products；Digestion products are attached；
（3）Convert, screen, detailed process is：
By step（2）In connection product convert to e. coli bl21 competent cell, carry out resistance screening；Picking is positive single
Bacterium colony, the identification of NdeI and BamHI double digestions is carried out after extracting plasmid；Identification is correctly recombinant expression carrier pET21b-
HEMBP（Pyr）.
3. solubility expression of protein can be promoted described in claim 1 and the recombinant expression carrier pET21b-HEMBP of expression quantity is improved
（Pyr）Application in protein expression, it is characterised in that for the prokaryotic expression system of Escherichia coli.
4. solubility expression of protein can be promoted as claimed in claim 3 and the recombinant expression carrier pET21b- of expression quantity is improved
HEMBP（Pyr）Application in protein expression, it is characterised in that be used to express list in the prokaryotic expression system of Escherichia coli
Clonal antibody Nb, PCV2VHHC3, antigen H5HA10, PCV2b cap, NLSFMD, HAFnt, FMDV98, polyprotein Cago60,
CdiGMP499（Caulobacter vibrioides）、CdiGMP026（Phenylobacterium zucineum）Or Prop
acid OAS。
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| CN111979257A (en) * | 2019-05-22 | 2020-11-24 | 上海凯赛生物技术股份有限公司 | Recombinant DNA and application thereof |
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| CN113355341A (en) * | 2021-05-13 | 2021-09-07 | 湖北省生物农药工程研究中心 | Fusion tag-based chitosanase expression promoting method and recombinant fusion chitosanase |
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| CN111979257A (en) * | 2019-05-22 | 2020-11-24 | 上海凯赛生物技术股份有限公司 | Recombinant DNA and application thereof |
| CN111978407A (en) * | 2019-05-22 | 2020-11-24 | 上海凯赛生物技术股份有限公司 | Heterologous expression method of lysine decarboxylase from thermophilic bacteria and application thereof |
| CN111979257B (en) * | 2019-05-22 | 2023-10-13 | 上海凯赛生物技术股份有限公司 | Recombinant DNA and application thereof |
| CN111978407B (en) * | 2019-05-22 | 2023-10-13 | 上海凯赛生物技术股份有限公司 | Heterologous expression method of lysine decarboxylase from thermophilic bacteria and application thereof |
| CN113355341A (en) * | 2021-05-13 | 2021-09-07 | 湖北省生物农药工程研究中心 | Fusion tag-based chitosanase expression promoting method and recombinant fusion chitosanase |
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