CN105950605A - Directional covalent immobilization method of biotin-protein ligases (BirA) on magnetic microsphere surface - Google Patents
Directional covalent immobilization method of biotin-protein ligases (BirA) on magnetic microsphere surface Download PDFInfo
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Abstract
The invention discloses a directional covalent immobilization method of biotin-protein ligases (BirA) on a magnetic microsphere surface. The method comprises the following steps: firstly, using a genetic engineering technology to express a biotin-protein ligase recombinant protein of which the N-terminal has an MLAQGS (methionine-leucine-alanine-glutamine-glycine-serine) sequence and a polyhistidine sequence; then utilizing the catalysis of transglutaminase, making -NH2 of glutamine in the biotin-protein ligase recombinant protein and -NH2 on the amino-modified magnetic microsphere surface form covalent connection, thus directionally immobilizing the biotin-protein ligases on the magnetic microsphere carrier surface by a manner of covalent binding. According to the invention, the new method for immobilizing BirA is established, so that the enzymatic catalysis activity of BirA can be fully kept, and can be repeatedly used to improve the use efficiency of BirA and lower the use cost.
Description
Technical field
The invention belongs to biochemical field, particularly relate to the enzyme of a kind of directional at-tachment, i.e. utilize biology side
Method by biotin ligase (BirA) with covalently bound mode directional at-tachment on Magnetic Microspheres-Carrier surface.
Background technology
Biotin ligase, also known as biotin-protein ligase, BirA enzyme, is by the 1 of escherichia coli BirA gene code
Individual bifunctional protein, this enzyme can produce inhibitory action as a kind of repressor protein to the operon of synthesizing biotinylated, even more important
Be that it can form biological acyl 5' adenylic acid by activated biotin, then biotin is transferred on biotin acceptor albumen.By
Adding 1 on destination protein can be by the peptide sequence (can biotinylated sequence, such as Avi-tag) of BirA enzyme identification, and BirA enzyme is i.e.
Can be attached on destination protein by biotin, labeling process is enzymatic reaction, more conventional chemical coupling techniques system
Standby protein-biotin conjugates is compared, and has efficiently, site-specific and the feature such as easy to operate.Biology due to BirA enzyme
Elementization ability, is widely used at protein labeling technical elements as a kind of important reagent, as protein purification, cell divide
Choosing, immunoassay etc..
BirA enzyme is colibacillary proper constituent, and natural expression is the lowest, expensive.BirA enzyme is applied to be catalyzed mesh
Protein biotinylation be under liquid environment, resolvase in reactant liquor neutralized reaction product together, after reaction enzyme can not reclaim weight
Multiple utilization, also makes the isolated and purified increasingly complex of product, therefore develops a kind of immobilized BirA enzyme, not only keep its zymetology
Catalysis activity, and reuse, improve the service efficiency of enzyme and reduce use cost, significant.Although enzyme is fixing
Change method has multiple, but the most the most frequently used fixing means is covalent coupling method, i.e. the nonessential group of pheron passes through covalency
Key and carrier form irreversible connection.But the higher structure of enzyme is the most sensitive to environment, physical factor, chemical factor and life
Thing factor all can make enzyme devitalization.The covalent bond immobilization behavior of chemically based method is by-the NH of pheron molecule2、-
COOH ,-OH or-SH isoreactivity group are combined, due to above-mentioned active group with surface of solid phase carriers active group generation covalent bond
Locational uncertainty in protein molecule and nonuniqueness, cause the binding pattern that this covalent bond is not fixed, when
Connection site affects the catalysis activity of enzyme when being positioned at enzyme active sites or neighbouring group.
Glutamine transaminage (Transglutaminase), can be with catalytic proteins intramolecular also known as T-5398
Crosslinking, intermolecular crosslinking, connection between protein and aminoacid and protein molecule in the hydrolysis of paddy ammonia phthalein amido,
Utilize this reaction by some limiting amino acids introducing protein to improve protein structure, thus the merit of protein can be improved
Can character and nutritive value thereof.The most microbe-derived glutamine transaminage (glutamine of microbe transaminase,
Microbial transglutaminase, is called for short MTG) it is widely used to the industries such as food.The machine of glutamine transaminage
Reason is the enzyme of catalyzing acyl transfer, using the γ-carboxamide groups of peptide chain GLN residue as acry radical donor.Acyl acceptor can
Being the epsilon-amino of lysine residue, primary amine groups or water in polypeptide chain, in acyl acceptor is polypeptide chain the ε of lysine residue-
During amino, with intermolecular connection in formation protein molecule；In acyl acceptor is polypeptide chain or the primary of other macromole
During amido, forming protein-protein or the moleculartie of protein-other macromole, principle is as follows.
R1—GluCONH2+NH2—R2→R1—GluCONH—R2+NH3 +
Summary of the invention
For above-mentioned prior art, it is an object of the invention to provide a kind of real by glutamine transaminage (MTG) catalysis
The existing BirA enzyme site-specific covalent bond fixing means on magnetic microsphere surface.The stereotaxis utilizing the present invention is fixed
Method, can realize the covalent coupling in BirA enzyme spcificity site, and keep the high catalytic activity of BirA enzyme.
For achieving the above object, the present invention adopts the following technical scheme that
First purpose of the present invention is to provide the site-specific covalency directional at-tachment side of a kind of biotin ligase
Method, comprises the following steps: first, utilizes technique for gene engineering to give expression to N end band MLAQGS (Met-Leu the-the third ammonia
Acid-glutamine-glycine-serine) the biotin ligase recombiant protein of sequence and polyhistidine sequence is (named
Gln-BirA recombiant protein)；Then utilize the catalytic action of glutamine transaminage, by the glutamine in Gln-BirA-
NH2-the NH of (donor) and amination modified magnetic microsphere surface2(receptor) is formed covalently bound, thus realizes utilizing biology side
Method by biotin ligase (BirA) with covalent bond mode directional at-tachment on Magnetic Microspheres-Carrier surface, can fully keep it
Zymetology catalysis activity, and reuse, improve the service efficiency of enzyme and reduce use cost.
Obtain through substantial amounts of recognition sequence is carried out screening, the present invention preferred MLAQGS (Met-Leu-the third
Propylhomoserin-glutamine-glycine-serine) sequence, further it is studied, obtain in N terminal modified MLAQGS sequence
Time, the enzymatic activity of obtained Gln-BirA recombiant protein is higher.
Preferably, described polyhistidine sequence is 6 polyhistidine sequence (6 × His sequences).
Preferably, described utilize technique for gene engineering to give expression to N end band at least to contain the biotin of consequent glutamine sequences even
The step connecing enzyme recombiant protein is as follows:
First, N end band MLAQGS (Met-Leu-Ala-Gln-Gly-serine) sequence is built
The recombinant expression carrier of the BirA enzyme recombiant protein of row and polyhistidine sequence, uses escherichia coli expression destination protein, so
Rear gained destination protein utilizes immobilization metal to chelate affinity chromatography (IMAC) Ni affinitive layer purification, obtains after purification
Gln-BirA recombiant protein.
It is further preferred that give expression to concretely comprising the following steps of Gln-BirA recombiant protein: utilize technique for gene engineering to build
Expression plasmid carrier pMLAQGS-His-BirA, E.coli BL21 (DE3) express Gln-BirA recombiant protein, and IMAC Ni is affine
Chromatography purification Gln-BirA recombiant protein.Through lot of experiments checking with analyze, use the E.coli BL21 (DE3) can efficient table
Reach Gln-BirA recombiant protein.
Preferably, BirA enzyme covalency, orientation are fastened on magnetic microsphere surface method particularly includes: utilize microorganism paddy
The catalytic action of glutamine transaminase, at the γ-carboxylic acyl of the glutamine (Q) of Gln-BirA recombinant protein N end MLAQGS sequence
Amido (donor) and-NH of amination modified magnetic microsphere surface2(receptor) forms covalent bond, thus by BirA enzyme covalency, fixed
To being fastened on magnetic microsphere surface.
Preferably, described BirA enzyme covalency, orientation are fastened on the concrete operation method on magnetic microsphere surface and are:
Amidized magnetic microsphere, biotin ligase recombiant protein (Gln-BirA recombiant protein) and glutamine are turned
Amine enzyme (MTG) hybrid reaction, collects reacted microsphere through Magneto separate after reaction, i.e. obtains covalently bound biotin ligase
Magnetic microsphere.
Further, the buffer system of above-mentioned reaction is PBS, pH7.5～8.5；The microsphere obtained after reaction is entered one
Step uses the PBS solution washing containing Tween-20, obtains the magnetic microsphere of covalently bound biotin ligase.
Further, described reaction temperature is 20～30 DEG C, and the response time is 5～7h.
Further, the hybrid reaction system of optimization is: in 1mL reaction system, containing 1mg amination magnetic microsphere (diameter
0.5～1.0 μm), 0.5mg recombinates BirA enzyme, 10U MTG, and reaction buffer system is the PBS (pH 8.0) of 20mM, 24 DEG C of concussions
Reaction 6h.Reacted microsphere is collected, with containing the PBS solution washing number that mass fraction is 0.01%Tween-20 through Magneto separate
Secondary, each 5min.The present invention obtains the reaction system of above-mentioned optimization through lot of experiments, makes the enzyme of the immobilized enzyme finally given
Vigor is higher.
Heretofore described 6 × His sequence is six histidine HHHHHH, is characterized in that molecular weight is little, does not changes
The biological structure of preserved duck egg white matter, does not change the dissolubility of protein, and it makes protein purification become extremely convenient, according to group ammonia
The principle that imidazole ring in acid can be combined with bivalent metal ion, utilizes metal ion affinity chromatography technology purification with His
The albumen of label, will be filled out by fixing bivalent metal ion (typically bivalence Ni ion) by the lysate containing destination protein
Material, the protein with 6*his label is i.e. combined with filler, and other albumen is not combined with filler, the most again with the miaow of high concentration
Destination protein can be eluted by azoles.6 × His sequence is through commonly used a kind of label in protein recombinant technique.
The definition of heretofore described recombinant expression carrier is: the exogenous nucleic acid sequences that can carry insertion enters in cell
Carry out the carrier expressed.
Heretofore described amination modified magnetic microsphere is that those skilled in the art's routine obtains, and is such as amino
Change the ferroso-ferric oxide microsphere modified.
Second object of the present invention is to provide a kind of immobilization biological element ligase using said method to prepare.
Third object of the present invention is to provide a kind of above-mentioned immobilization biological element ligase as protein labeling
Application in reagent.Main application direction is protein purification, cell sorting, immunoassay etc..The invention has the beneficial effects as follows:
(1) immobilization BirA enzyme provided by the present invention, is to utilize biological method to realize at the specific site of BirA enzyme
Covalently bound directional at-tachment, it is to avoid the random coupling of chemical covalent combined techniques, be conducive to keeping fixing after BirA enzyme catalysis
Activity.A kind of BirA enzyme immobilizatio new method that the present invention sets up, can fully keep its zymetology catalysis activity, and repeat to make
With, improve the service efficiency of enzyme and reduce use cost.
(2) carrier plays pivotal role in enzyme immobilization technology, so selecting properly during immobilized enzyme
Carrier extremely important.The present invention is by screening variety carrier, and result obtains use amination magnetic microsphere can be real
The covalent coupling that existing BirA enzyme is good, it is possible to quick and enzyme reaction, not only the fixed amount of enzyme is higher, and enzyme activity is good.
(3) present invention is obtained by technique for gene engineering through substantial amounts of experiment and gives expression to N end band MLAQGS sequence and many
The biotin ligase recombiant protein (named Gln-BirA recombiant protein) of polyhistidine sequence, its immobilized enzyme formed is not
Only enzyme fixed amount (albumen coupling amount) is higher, and its enzymatic activity is the highest.
Accompanying drawing explanation
Fig. 1 is the N-end restructuring Gln-BirA structural representation with MLAQGS sequence.
Fig. 2 is that the transaminase-catalyzed amination magnetic microsphere surface site specificity of glutamine of microbe is covalently bound solid
Fixed restructuring Gln-BirA schematic diagram.
Fig. 3 is that immobilization BirA enzyme recycles and property is to enzymatic activity.
Detailed description of the invention
Embodiment 1, the structure of restructuring MLAQGS-His-BirA prokaryotic expression vector
BirA gene according to Escherichia coli K-12MG1655 (No:EG10123) logged in GenBank
Sequence, designs following primer:
Primer 1, as shown in SEQ ID No:1:
5′-GAATTCGATGCTAGCGCAGGGATCACACCATCACCATCACCATATGAAGGATAACA-3′；
Primer 2, as shown in SEQ ID No:2:
5′-CCGGGATCCTTATTTTTCTGCACTACGCAGGG-3′
Wherein, Primer 1 is containing EcoR I restriction enzyme site, MLAQGS sequence, 6 × His sequence, and Primer 2 contains
BamH I restriction enzyme site.
With E.coli DH5 α genome as template, Primer 1 and Primer 2 expands BirA base for primer PCR mode
Cause, the genetic fragment obtained is cloned into pUC18 plasmid EcoR I/BamH I restriction enzyme site according to molecular biology method, obtains
Obtain pMLAQGS-His-BirA expression plasmid carrier.
Embodiment 2, the structure of restructuring BirA-His-MLAQGS prokaryotic expression vector
BirA gene according to Escherichia coli K-12MG1655 (No:EG10123) logged in GenBank
Sequence, designs following primer:
Primer 3, as shown in SEQ ID No:3:
5′-GAATTCGATGAAGGATAACA-3′；
Primer 4, as shown in SEQ ID No:4:
5′-CCGGGATCCCTGGTGCTGGTGCTGGTGTGATCCCTGCGCTAGCATTTATTTTTCTGCACTACGCAGGG-3′
Wherein, Primer 3 containing EcoR I restriction enzyme site, Primer 4 containing 6 × His sequence, MLAQGS sequence and
BamH I restriction enzyme site.
With E.coli DH5 α genome as template, Primer 3 and Primer 4 expands BirA base for primer PCR mode
Cause, the genetic fragment obtained is cloned into pUC18 plasmid EcoR I/BamH I restriction enzyme site according to molecular biology method, obtains
Obtain pBirA-His-MLAQGS expression plasmid carrier.
Embodiment 3, the technique for gene engineering preparation of restructuring MLAQGS-His-BirA and BirA-His-MLAQGS and purification
PMLAQGS-His-BirA plasmid CaCl2Method Transformed E .coli BL21 (DE3), builds pMLAQGS-His-BirA/
BL21 engineering bacteria.
PBirA-His-MLAQGS plasmid CaCl2Method Transformed E .coli BL21 (DE3), builds pBirA-His-MLAQGS/
BL21 engineering bacteria.
Above two engineering bacteria activated overnight, transfers in fresh LB culture medium (ampicillin concentration by 5% inoculum concentration
100 μ g/mL), 37 DEG C of shaken cultivation 12-16h, centrifugal collection thalline.With containing 20mM imidazoles, the PBS solution of 500mM NaCl
(20mM, pH 8.0) resuspended thalline, ultrasonication, 10000rpm centrifugation bacterial chip, after supernatant 0.22 μm membrane filtration,
0.5mL/min circulates HisTrap chromatographic column, rinses chromatographic column to A with the PBS solution containing 20mM imidazoles, 500mM NaCl280Value
No longer change.With containing 250mM imidazoles, the PBS eluting of 500mM NaCl, collect eluent, Millipore ultra-filtration centrifuge tube
(10kD) 5000rpm is centrifuged desalination, and washs, is concentrated into suitable volumes with PBS solution (20mM, pH 8.0), through 12%SDS-
PAGE analyzes, and destination protein relative molecular weight is 36.7kD, is consistent with theoretical value.Gel gray scale scanning result display purification
The purity of MLAQGS-His-BirA recombiant protein and BirA-His-MLAQGS recombiant protein is more than 96%.To contain at N-end
There is the MLAQGS-His-BirA albumen named restructuring Gln-BirA enzyme of MLAQGS sequence, contain MLAQGS sequence at C-end
BirA-His-MLAQGS albumen named restructuring BirA-Gln enzyme.
Embodiment 4, restructuring Gln-BirA enzyme and the restructuring BirA-Gln enzyme directional at-tachment on amination magnetic microsphere surface
Utilize the catalytic action of glutamine of microbe transaminase, at the MLAQGS tag peptide sequence of BirA recombiant protein
Glutamine (Q) and-NH of amination modified magnetic microsphere surface2Form covalent bond, thus BirA enzyme covalency, orientation are connected
It is fixed on magnetic microsphere surface.The reaction system optimized is as follows:
In 1mL reaction system, containing 1mg amination magnetic microsphere (diameter 0.5～1.0 μm), 0.5mg restructuring BirA enzyme (weight
Group Gln-BirA enzyme or restructuring BirA-Gln enzyme), 10U MTG, reaction buffer system is the PBS (pH8.0) of 20mM, 24 DEG C of concussions
Reaction 6h.Collect reacted microsphere through Magneto separate, wash 3 times with containing the PBS solution that mass fraction is 0.01%Tween-20,
5min every time.
Through BCA method determining the protein quantity analyze, every mg magnetic microsphere surface can coupling 0.06mg restructuring Gln-BirA enzyme or
Restructuring BirA-Gln enzyme, for 1.6pmol enzyme/cm2Microsphere, albumen coupling amount higher than document report 0.66pmol alkali phosphatase/
cm2Microsphere [K.Moriyama, K, Sung, M.Goto, N.Kamiya, Immobilization of alkaline
phosphatase on magnetic particles by site-specific and covalent cross-linking
catalyzed by microbial transglutaminase,J.Biosci.Bioeng.111(2011):650–653]。
Amination Magnetic Microspheres-Carrier in the present embodiment is replaced with amination agarose microbeads, amination polystyrene
Microsphere, amino chitin microsphere, the reacted microsphere of separated collection, use BCA method determining the protein quantity to analyze, consolidating of its enzyme
Quantitatively less than 1.6pmol enzyme/cm2Microsphere.
Embodiment 5, restructuring Gln-BirA enzyme or the enzyme activity analysis of restructuring BirA-Gln enzyme
With containing 15 amino acid whose can the ZZ-Avitag recombiant protein of biotinylation tag (Avitag label)
(18.3kD) it is substrate, analyzes the biotinylation ability of Gln-BirA enzyme substrate for enzymatic activity albumen；4'-hydroxyazobenzene-2-carboxylic
Acid (HABA) method [Hirsch JD, Haugland RP, Conjugation of antibodies to biotin, Methods
Mol.Biol.295 (2005) 135 154.] measure the biotin number being marked at ZZ-Avitag albumen.
Reaction system: cumulative volume 50 μ L, containing 10 μ g restructuring Gln-BirA enzymes or restructuring BirA-Gln enzyme, 38 μMs of ZZ-
Avitag recombiant protein, and 50mM N, N-bicine N-(pH8.3), 10mM ATP, 10mM magnesium acetate, 50mM D-is raw
Thing element.Above-mentioned reactant liquor under the conditions of 30 DEG C, catalytic reaction 30min.The life being connected to ZZ-Avitag albumen is measured through HABA method
Thing prime number mesh, the enzyme activity recording restructuring Gln-BirA enzyme is 4600U/mg pheron, the relative of BirA-Gln enzyme of recombinating
Enzyme activity is 3900U/mg pheron.Result shows to introduce MLAQGS-His sequence gained restructuring Gln-in the restructuring of BirA enzyme N-end
The enzyme activity of BirA enzyme is significantly higher than the enzyme activity of His-MLAQGS sequence gained restructuring BirA-Gln enzyme.
Enzyme activity unit defines: under the conditions of 30 DEG C, by containing the 1pmol in 38 μMs of peptide substrate reactant liquors in 30min
Needed for peptide substrate biotinylation, enzyme amount is defined as 1 enzyme unit (U).
Embodiment 6, oriented immobilization restructuring Gln-BirA enzyme and the enzyme activity analysis of restructuring BirA-Gln enzyme
With ZZ-Avitag recombiant protein as substrate, analyze the directional at-tachment MLAQGS-His-BirA on magnetic microsphere surface
Or the biotinylation ability of BirA-His-MLAQGS recombinase catalytic substrate albumen；4'-hydroxyazobenzene-2-carboxylic acid (HABA)
Method measures the biotin number being marked at ZZ-Avitag albumen.
Reaction system: cumulative volume 50 μ L, containing 100 μ g magnetic microspheres (coupling 10 μ g recombinase), 38 μMs of ZZ-Avitag weights
Histone, and 50mM N, N-bicine N-(pH8.3), 10mM ATP, 10mM magnesium acetate, 50mM Bio.On
State reactant liquor under the conditions of 30 DEG C, catalytic reaction 30min.Through Magneto separate magnetic microsphere, HABA method measures and is connected to ZZ-Avitag
The biotin number of albumen.
Result shows, the enzyme activity recording immobilization restructuring Gln-BirA enzyme is 4630U/mg pheron, relative enzyme
Vigor is 94.5% before fixing；The enzyme activity of immobilization restructuring BirA-Gln enzyme is 3130U/mg pheron, relative enzyme
Vigor is 80.3% before fixing.Show that the restructuring Gln-BirA enzyme after immobilization preferably maintains the catalysis activity of enzyme, can
See, express compared to C end band MLAQGS sequence and polyhistidine sequence, N end band MLAQGS sequence and polyhistidine sequence
Afterwards the three dimensional structure of biotin ligase is affected less.
By above-mentioned 50 μ L reaction system and conditions, with the magnetic microsphere of a collection of coupling restructuring Gln-BirA enzyme, circulation catalysis
38 μMs of ZZ-Avitag protein 10s, HABA method measures the biotinylation effect of ZZ-Avitag albumen.Every secondary response is complete, warp
Magneto separate, the magnetic microsphere PBS solution washing containing 0.01%Tween-20 3 times, each 5min, for next time.Result
Display is used for multiple times, and fixing BirA enzyme still maintains higher enzymatic activity, and the enzyme activity that the 10th time uses maintains the
The 92.6 ± 6.1% of nonrecoverable enzyme activity.
The amination Magnetic Microspheres-Carrier that immobilization in the present embodiment is recombinated in Gln-BirA enzyme is replaced with amination
Agarose microbeads, amination polystyrene microsphere, amino chitin microsphere, record the relative enzyme of immobilization restructuring Gln-BirA enzyme
Vigor, its enzyme activity and the present invention are with relatively low compared with the immobilized enzyme of amination Magnetic Microspheres-Carrier, therefore the present invention is excellent
Choosing is with amination magnetic microsphere as carrier.
Claims (10)
1. a site-specific covalency directional at-tachment method for biotin ligase, is characterized in that, comprise the following steps: first
First, technique for gene engineering is utilized to give expression to N end band Met-Leu-Ala-Gln-Gly-serine sequence
Row and the biotin ligase recombiant protein of polyhistidine sequence；Then, utilize the catalytic action of glutamine transaminage, will
-the NH of the glutamine in described biotin ligase recombiant protein2-NH with amination modified magnetic microsphere surface2Formed altogether
Valency connect, thus realize by biotin ligase with covalent bond mode directional at-tachment on Magnetic Microspheres-Carrier surface.
2. the method for claim 1, is characterized in that: described polyhistidine sequence is 6 polyhistidine sequence.
3. the method for claim 1, is characterized in that: described utilize technique for gene engineering to give expression to N end band at least to contain
The step of the biotin ligase recombiant protein of consequent glutamine sequences is as follows:
N end band Met-Leu-Ala-Gln-Gly-silk ammonia is built first with technique for gene engineering
The prokaryotic expression carrier of the biotin ligase recombiant protein of acid sequence and 6 × His sequence, then uses escherichia coli expression institute
State biotin ligase recombiant protein.
4. method as claimed in claim 3, is characterized in that: described utilize technique for gene engineering to give expression to N end band at least to contain
Specifically comprising the following steps that first with technique for gene engineering construction expression of the biotin ligase recombiant protein of consequent glutamine sequences
Plasmid vector pMLAQGS-His-BirA, then uses E.coli BL21 (DE3) to express described biotin ligase restructuring egg
In vain.
5. the method as described in claim 3 or 4, is characterized in that: by biotin ligase weight described in described escherichia coli expression
After histone, then use immobilization metal to chelate affinity chromatography Ni affinitive layer purification, obtain Gln-BirA weight after purification
Histone.
6. the method for claim 1, is characterized in that: biotin ligase covalency, orientation are fastened on magnetic microsphere
Surface method particularly includes: utilize the catalytic action of glutamine of microbe transaminase, at biotin ligase recombinant protein N end
γ-the carboxamide groups of the glutamine of Met-Leu-Ala-Gln-Gly-lysine sequence and ammonia
-the NH of base modified magnetic microsphere surface2Form covalent bond, thus BirA enzyme covalency, orientation are fastened on magnetic microsphere
Surface.
7. method as claimed in claim 6, is characterized in that: biotin ligase covalency, orientation are fastened on magnetic microsphere
The concrete operation step on surface is: by amidized magnetic microsphere, biotin ligase recombiant protein and glutamine transaminage
Hybrid reaction, collects reacted microsphere through Magneto separate after reaction, and the magnetic i.e. obtaining covalently bound biotin ligase is micro-
Ball.
8. method as claimed in claim 7, is characterized in that: described reaction temperature is 20～30 DEG C, and the response time is 5～7h；
Preferably, hybrid reaction system is: in 1mL reaction system, and containing 1mg amination magnetic microsphere, 0.5mg recombinates BirA enzyme, 10U
MTG, reaction buffer system is the PBS of 20mM, 24 DEG C of concussion reaction 6h, collects reacted microsphere through Magneto separate, with containing quality
Mark is that the PBS solution of 0.01%Tween-20 is washed for several times；I.e. can get the magnetic microsphere of covalently bound biotin ligase.
9. the immobilization biological element ligase that method any one of claim 1～8 prepares.
10. the immobilization biological element ligase described in claim 9 is as the application in protein labeling reagent.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106978429A (en) * | 2017-03-16 | 2017-07-25 | 中国人民解放军第四军医大学 | A kind of bovine enterokinase light chain load magnetic bead and its preparation method and application |
| CN107190028A (en) * | 2017-05-25 | 2017-09-22 | 高永华 | The extracting method of forulic acid in a kind of rice bran meal |
| CN116515799A (en) * | 2022-04-06 | 2023-08-01 | 无锡佰翱得生物科学股份有限公司 | Recombinant Sortase A enzyme and immobilization method and application thereof |
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| CHANG-MEI YU ET AL: "Site-specific, covalent immobilization of BirA by microbialtransglutaminase: A reusable biocatalyst for in vitro biotinylation", 《ANALYTICAL BIOCHEMISTRY》 * |
| KOUSUKE MORIYAMA ET AL: "Immobilization of alkaline phosphatase on magnetic particles by site-specific and covalent cross-linking catalyzed by microbial transglutaminase", 《JOURNAL OF BIOSCIENCE AND BIOENGINEERING》 * |
| NORIHO KAMIYA ET AL: "Functional Immobilization of Recombinant Alkaline Phosphatases Bearing a Glutamyl Donor Substrate Peptide of Microbial Transglutaminase", 《JOURNAL OF BIOSCIENCE AND BIOENGINEERING》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106978429A (en) * | 2017-03-16 | 2017-07-25 | 中国人民解放军第四军医大学 | A kind of bovine enterokinase light chain load magnetic bead and its preparation method and application |
| CN106978429B (en) * | 2017-03-16 | 2019-11-15 | 中国人民解放军第四军医大学 | A kind of bovine enterokinase light chain load magnetic bead and its preparation method and application |
| CN107190028A (en) * | 2017-05-25 | 2017-09-22 | 高永华 | The extracting method of forulic acid in a kind of rice bran meal |
| CN107190028B (en) * | 2017-05-25 | 2019-01-15 | 西安海斯夫生物科技有限公司 | The extracting method of ferulic acid in a kind of rice bran meal |
| CN116515799A (en) * | 2022-04-06 | 2023-08-01 | 无锡佰翱得生物科学股份有限公司 | Recombinant Sortase A enzyme and immobilization method and application thereof |
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