CN106317227A - Affinity ligand, construction method, affinity chromatography medium, preparation method and application - Google Patents
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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Abstract
The invention provides an affinity ligand, a construction method, an affinity chromatography medium, a preparation method and application. The affinity ligand is constructed on the basis that the structure of NpuDnaE-N, namely, IN is transformed, wherein cysteine is additionally arranged at the C terminal of IN to be used for being covalently bonded with chromatography microspheres, mutated Cys1Ala is introduced into a first amino acid located at the N terminal of natural IN so as to avoid a fracture reaction of the N terminal, and His-tag is arranged at the N terminal of an IN segment to be used for purifying a ligand segment. Accordingly, construction is completed by depending on the fracture reaction of intein, and therefore not only is the cost reduced, but also contamination to target protein can be prevented; the fracture rate of 3h is still high in the presence of a reducing agent, and the constructed affinity purification medium has the good purification effect and the high repeated utilization rate.
Description
Technical Field
The invention belongs to the field of protein purification, and relates to a modified NpuDnaE-N-terminal splicing structural domain (I)N) The formed affinity ligand and a chromatography medium for protein affinity purification are constructed by covalently crosslinking the C end of the affinity ligand and chromatography microspheres. The target protein only needs to be constructed on the molecular level ICC terminal of (A) andCfusion expression by in vitro ICAnd INThe target protein can be purified by affinity binding and condition-induced fragmentation control.
Background
The application of the purification tag greatly simplifies the process of recombinant protein purification, and has immeasurable application value to modern biotechnology. However, the affinity purification tag introduced in the target protein must be removed by treatment with a proteolytic or chemical cleavage reagent. Therefore, in large-scale production, the problem of removing the purification tag restricts the wide application of the purification tag. To solve this problem, an intein-mediated spontaneous fragmentation purification tag without the use of proteases was invented.
Inteins are a segment of polypeptide chain in a precursor immature protein, and can be cut from the precursor protein through a series of autocatalytic reactions such as rearrangement, transesterification and cyclization, and the protein polypeptide chains at two ends (protein exteins) are connected through a natural peptide bond. The split intein is structurally N-terminal (I)N) And a C-terminal region (I)C) An intein isolated from each other. When I isNAnd ICAfter the two fragments are connected and the structure is restored, splicing of the two-end exteins can be completed according to a standard intein splicing approach. The splicing activity of the split intein is via INAnd ICThe two have strong mutual recognition capability, which is the basis of the application of intein in an affinity purification system.
In general, inteins spontaneously undergo N-/C-terminal cleavage in the presence of a reducing agent, cleaving the target protein at one end. Since this series of reactions can proceed spontaneously in vitro without the action of enzymes, purification of protein expression by cleavage of intein pathway can remove purification tags as well as purification efficiently.
Although the cleavage of inteins is certainly worthwhile in protein purification, there are some disadvantages in their use. First, INIs immobilized to the chromatographic microspheres by affinity adsorption between the purification tag and the medium upon a change in solution conditions(pH, temperature or reducing agent), the affinity tag is likely to fall off from the medium, and secondary pollution of the target protein is caused; in addition, there are problems with the rate of cleavage reaction of inteins and with the control of the rate of cleavage reaction. The cleavage reaction rate of some inteins is slow (more than 12 h), which prolongs the purification process and reduces the purification efficiency. However, effective inhibition of the cleavage reaction by rapidly cleaved inteins such as NpuDnaE is problematic, which may result in loss of the target protein during the process of washing away the foreign proteins, and thus, decrease in purification efficiency.
By study I, Zhilei Chen et alNAnd ICThe mode of interaction between them has found that steric hindrance between the target protein and the mediator may affect the cleavage activity of the cleaved intein. According to the principle of steric hindrance, the experimental studies of the present invention have found that the purification of the affinity ligand fragment INAnd ICThe location of the His-tag of the fusion expression fragment with the protein of interest also affects the rate of the cleavage reaction. For the cleavage intein-mediated affinity purification system, the key to improving the purification efficiency of the system is not only to increase the rate of the cleavage reaction, but also to inhibit the cleavage reaction during the loading and washing of the hetero-protein. It was found that Zn is present only when the first amino acid of NpuDnaE is Cys2+Can bind to a specific site in the intein NpuDnaE to inhibit the cleavage reaction. Therefore, the invention adds Zn in the stages of loading and washing the heteroprotein without mutating the first amino acid of the NpuDnaE2+Effectively inhibits the premature fracture, thereby improving the purification efficiency of the system.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above and/or other problems associated with the prior art needle affinity ligands and uses thereof.
Accordingly, it is an object of the present invention to provide an N-terminal splicing domain of an engineered NpuDnaE (I)N) The affinity ligand formed.
To solve the above technical problem, according to an aspect of the present invention, the present invention provides the following technical solutions: affinity ligand recombination IN *Proteins, said affinity ligands being constructed on the protein of npudnaE-N, native INThe structure of the device is modified; wherein, natural INThe C end of the chromatographic microsphere is added with cysteine for covalent binding with the chromatographic microsphere; natural INThe first amino acid of the N end is introduced into mutation Cys1Ala, so that the occurrence of N end breakage reaction is avoided; his-tag placed in nature INThe N-terminus of the fragment was used to purify the ligand fragment.
As a preferred embodiment of the affinity ligand according to the invention, wherein: the natural NpuDnaE gene sequence template is shown in a sequence table SEQ ID No. 3.
As a preferred embodiment of the affinity ligand according to the invention, wherein: the His-tag is placed in the natural INN-terminal of the fragment, mutant Cys1Ala, native INThe C end of the protein is added with cysteine, the gene sequence of the cysteine is shown as a sequence table SEQ ID No.4, and the protein sequence of the cysteine is shown as a sequence table SEQ ID No. 6.
Another object of the present invention is to provide a method for constructing an affinity ligand.
In order to solve the above technical problem, according to another aspect of the present invention, the present invention provides the following technical solutions: carrying out molecular modification on the N-terminal splicing structural domain of natural fractured intein NpuDnaE to obtain modified INThe fragment is named as IN *Including, in nature INAdding a Cys to the C-terminal of (1); in INAdding a His-tag sequence at the N end of the fragment; adding Nde I and Hind III enzyme cutting sites into the primerPointing and using it to point IN *The fragment is inserted into an expression vector pET28a, and the obtained protein is the affinity ligand fragment.
As a preferred embodiment of the method for constructing an affinity ligand according to the present invention, wherein: the primer, wherein, IN *The gene sequences of the upstream primer and the downstream primer with modified sequences are respectively shown as SEQ ID No.1 and SEQ ID No.2 of the sequence table.
As a preferred embodiment of the method for constructing an affinity ligand according to the present invention, wherein: further comprises the construction of recombinant bacteria, PCR is carried out by taking pRSFDuet-1-NpuDnaE plasmid as a template to obtain a modified fragment IN *The recombinant plasmid pMD19-T-I is constructed by utilizing the vector pMD19-TN *Coli JM109 was transformed to be competent, and then the constructed I was cleaved with Nde I and Hind III enzymesN *The sequence is connected with expression plasmid pET-21b by enzyme digestion to obtain recombinant expression plasmid pET-21b-IN *Finally, competent E.coli BL21(DE3) was transformed to obtain the modified recombinant IN *Expression strain E.coli BL21(DE3)/pET-21b-IN *；NpuDnaEIN *The recombinant bacterium E.coli BL21(DE3)/pET-21b-IN *Carrying out induced expression to obtain the modified recombinant IN *A protein.
As a preferred embodiment of the method for constructing an affinity ligand according to the present invention, wherein: and recombinant IN *Protein pairing ICThe gene sequence and the protein sequence of the GFP fragment are shown in the sequence tables SEQ ID No.5 and SEQ ID No. 7.
It is a further object of the present invention to provide an affinity chromatography medium obtained by coupling with the above-mentioned affinity ligands.
To solve the above technical problem, according to still another aspect of the present invention, the present invention provides the following technical solutions: an affinity chromatographic medium prepared from affinity ligand through coupling reactionN *The fragments are affinity ligands, by activationCovalently coupling the spacer to the agarose microspheres to form pair ICThe affinity chromatography medium with fusion protein affinity has the structure of agarose microsphere-activated middle arm-IN *Affinity ligand:
the fourth object of the present invention is to provide a method for obtaining an affinity chromatography medium by coupling with the above-mentioned affinity ligands.
To solve the above technical problem, according to a fourth aspect of the present invention, the present invention provides the following technical solutions: a coupling preparation method by utilizing affinity ligand, as shown in the specificationN *The fragment is affinity ligand and is covalently coupled to agarose microspheres to form a pair ICAn affinity chromatography medium with fusion protein having affinity effect comprises activating agarose containing hydroxyl group with diepoxide 1, 4-butanediol diglycidyl ether to obtain activated epoxy agarose containing a long hydrophilic chain; the ethylene oxide groups on the activated epoxy agarose and IN *The sulfhydryl of the fragment C-terminal Cys reacts to prepare the affinity ligand fragment IN *Coupling with activated agarose medium to obtain affinity chromatography medium.
The invention also aims to provide an application of the affinity chromatography medium, which is to pack the affinity chromatography medium into a column; with a Zn content of 1mmol/L2+The PBS buffer solution is used for balancing; will contain the protein of interest ICLoading the crude fusion protein sample; with no Zn content2+Washing with PBS buffer solution; washing the chromatography medium with a fragmentation buffer solution containing 50mmol/L DTT, and standing at room temperature for 3 h; eluting the fractured target protein by using an elution buffer solution and collecting; i isN *Regenerating the affinity chromatographic column.
The invention has the following beneficial effects: the invention is completed by means of the self-cleavage reaction of the intein, thereby not only reducing the cost, but also preventing the pollution to the target protein; the invention has high breakage rate for 3h in the presence of a reducing agent, and the affinity purification medium constructed by the invention has good purification effect and high reutilization rate.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a diagram illustrating a system for affinity purification of a split intein NpuDnaE-N-terminal affinity ligand of the present invention and its components;
FIG. 2 shows the present invention IN *Flow chart for affinity purification of affinity chromatography media, wherein ① represents preparation of affinity chromatography media, ② represents loading, ③ represents washing of hetero-proteins, ④ represents induction of fragmentation and elution, and ⑤ represents regeneration of affinity chromatography media;
FIG. 3 shows the present invention IN *A process diagram of a target protein purification process by an affinity chromatography column is shown, wherein 1 represents loading, and 2 represents no Zn2+Washing with Buffer A, wherein 3 represents adding a fragmentation Buffer and maintaining for 5 hours, and 4 represents eluting with an elution Buffer;
FIG. 4 shows the present invention IN *SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) verification graph of purification efficiency of target protein by using affinity chromatography column, wherein 1 represents protein Marker, and 2 represents IC-a coarse sample of GFP fragments, 3 for the sample permeate at stage a in fig. 3, 4 for the sample at stage B in fig. 3, 5 for the elution sample at stage C in fig. 3, and 6 for the Buffer D elution sample;
FIG. 5 shows the present invention IN *A line graph of the recycling efficiency of the affinity chromatography column.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described herein, and it will be readily apparent to those of ordinary skill in the art that the present invention may be practiced without departing from the spirit and scope of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The invention designs an N-terminal splicing structural domain (I) of modified NpuDnaEN *) The formed affinity ligand is covalently cross-linked with chromatography microspheres through the C end to construct a novel chromatography medium for protein affinity purification. The target protein only needs to be constructed on the molecular level ICAnd with I at the C-terminus (corresponding to the role of an affinity tag)CFusion expression by in vitro ICAnd INThe purification of the target protein can be realized by affinity binding and splicing reaction under the control of condition induction.
The affinity ligand fragment was constructed on the fragment of npudnaE-N (I)N) The structure of the device is improved. In INThe C end of the chromatographic microsphere is added with cysteine for covalent binding with the chromatographic microsphere; in nature INThe first amino acid of the N end is introduced into mutation Cys1Ala, so that the occurrence of N end breakage reaction is avoided; his-tag for ligand fragment purification was placed in INThe N-terminus of the fragment.
The construction method of the modified NpuDnaE N-terminal splicing structural domain affinity ligand comprises the following steps:
the method comprises the following steps: primer design
The invention carries out molecular modification on the N-terminal splicing structural domain of the natural fractured intein NpuDnaE. Modified INThe fragment is named as IN *The content of the modification is in the natural INAdding a Cys to the C-terminal of (1), and adding a Cys to the C-terminal of (1)NHis-tag sequence is added to the N-terminal of the fragment. Since the modified sites are located at both ends of the target fragment, pair I can be directly completed by designing primersNAnd (5) modifying the fragment. Two enzyme cutting sites of Nde I and Hind III are added to the primer, and I is cut by using the enzyme cutting sitesN *The fragment was inserted into the expression vector pET21b, and the protein obtained by expression in this way was the desired fragment of the affinity ligand.
Step two: construction of recombinant bacterium
The invention carries out PCR by taking pRSFDuet-1-NpuDnaE plasmid as a template according to the primer designed in the step one to obtain modified INFragment IN *The recombinant plasmid pMD19-T-I is constructed by utilizing the vector pMD19-TN *Coli jm109 for glycerol frozen tube storage. Then utilizing Nde I and Hind III two enzyme cutting sites to construct the constructed IN *The sequence is connected with expression plasmid pET-21b by enzyme digestion to obtain recombinant expression plasmid pET-21b-IN *Finally, transformation of competent E.coli BL21(DE3) to obtain the modified recombinant IN *Expression strain E.coli BL21(DE3)/pET-21b-IN *Preparing a glycerol freezing tube for preservation and standby;
step three: NpuDnaEIN *Expression purification of
The recombinant bacterium E. coli BL21(DE3)/pET-21b-I constructed in the step (2) is treated by IPTGN *Carrying out induced expression to obtain the modified recombinant IN *A protein. Obtaining purified I by using Ni affinity chromatography columnN *For crosslinking with chromatography microspheres.
Step four: i isN *Preparation of affinity chromatography media as ligands
Activated epoxy agarose containing a long hydrophilic chain can be obtained by activating agarose containing hydroxyl groups using the diepoxide 1, 4-butanediol diglycidyl ether (BDCE). Activating the oxirane groups on agarose with IN *The sulfhydryl (-SH) of the Cys at the C end of the fragment reacts to prepare the affinity ligand fragment IN *Coupling with activated agarose medium to obtain pair ICThe fusion protein has affinity chromatography medium with affinity effect.
(1) Primer design
Example 1
Using PCR technology to carry out molecular modification on the N-terminal splicing structural domain of natural fractured intein NpuDnaE, and carrying out modification on INThe fragment is named as IN *. Design I according to the Gene sequence supplied by GeneBank, using the molecular biology software PrimerPremier 5N *Forward primer S '-N and reverse primer a' -N:
S’-N：GGAATTCCATATGCATCATCATCATCATCACGCATTAAGCTATGAAACGGAAA
A’-N：CCCAAGCTTTTAACAATTCGGCAAATTATCAAC
nde I enzyme cutting site and Hind III enzyme cutting site are respectively introduced into the forward primer and the reverse primer, wherein the inclined part is the enzyme cutting site, and the S' -N underlined part is an added His tag and INMutation of the first amino acid Cys1Ala, with a-N underlined, is an added cysteine and a stop codon. The designed primers were synthesized by Biotechnology engineering (Shanghai) GmbH.
(2) Recombinant vector pMD19-T-IN *Construction of
Example 2
Plasmid pRSFDuet-1-NpuDnaE was extracted using S '-N, A' -N as a primer and plasmid pRSFDuet-1-NpuDnaE as a template, and ExTaq DNA polymerase for IN *The sequences were subjected to PCR. The 50. mu.L system was selected according to the ExTaq DNA polymerase protocol.
And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; performing denaturation at 94 ℃ for 30s, annealing at 46 ℃ for 30s, and extension at 72 ℃ for 30s, and performing 30 cycles; finally, the temperature is kept at 72 ℃ for 10 min.
Recovery of I Using agarose gel recovery kitN *Fragmenting to obtain I with an adenine at each endN *Sequence, base complementary pairing principle is utilized to pair IN *The sequence is connected to a T vector pMD19-T to obtain a recombinant plasmid pMD19-T-IN *. Transforming the recombinant plasmid into competent cell E.coli JM109 to obtain recombinant strain E.coli JM109/pMD19-T-IN *And (5) preserving the mixture in a glycerol freezing tube for later use.
(3) Recombinant vector pET-21b-IN *Construction of
Example 3
Coli JM109/pMD19-T-I strainN *The plasmid was extracted, and then pMD19-T-I was prepared using restriction enzymes Nde I and Hind IIIN *Carrying out enzyme digestion, recovering the product gel of the enzyme digestion, and then connecting to obtain a recombinant expression plasmid pET-21b-IN *Finally, competent E.coli BL21(DE3) was transformed to obtain the modified recombinant IN *Expression strain E.coli BL21(DE3)/pET-21b-IN *And preparing a glycerol freezing tube for preservation and standby.
(4)NpuDnaEIN *Purification of expression and IN *Preparation of affinity chromatography column
Example 4
The recombinant bacterium E.coli BL21(DE3)/pET-21b-I obtained in example 3 was usedN *Overnight culture, transfer to 50mL LB medium, culture to OD600When the concentration was 0.6, IPTG was added to the final concentration of 0.1M, and expression was induced at 16 ℃ and 180 rpm. The cultured cells were collected by centrifugation, and the supernatant was collected by ultrasonication. The supernatant was applied to a Ni affinity chromatography column previously equilibrated with an equilibration buffer (20mM imidazole; 0.5M NaCl; 10mM Tris-HCl; pH 8.0) which eluted 7-8 column volumes to equilibrium, followed by elution with an elution buffer (250mM imidazole; 0.5M NaCl; 10mM Tris-HCl; pH 8.0). Collecting eluted IN *The concentration was determined by SDS-PAGE. Purification of the expressed affinity ligand fragment I by means of Ni affinity chromatography packingN *And freeze-drying the purified product for later use.
Example 5
Activated epoxy agarose containing a hydrophilic chain can be obtained by activating agarose 6FF containing hydroxyl groups with 1, 4-butanediol and glycidyl ether (BDEC). Equal amount of agarose microspheres are mixed with 1, 4-butanediol and glycidyl ether, and equal amount of 0.6mol/L NaOH is added, at 30 ℃, 200r/min, 10 h. After the reaction is finished, the activated agarose microspheres are washed by 20% ethanol and a large amount of deionized water for later use.
Example 6
With coupling buffer (0.1mol/L Na)2CO3/NaHCO3pH 10.0) solubilisation of the affinity ligand fragment IN *Coupled with epoxy activated agarose preparation IN *An affinity chromatography medium.The coupling conditions were: 40 ℃, 150r/min and 16 h. The coupled affinity medium is washed by deionized water and 20% ethanol, is dried by suction through a Buchner funnel, and is stored in 20% ethanol at 4 ℃.
With IN *The fragment is affinity ligand and is covalently coupled to agarose microspheres through activating a middle arm to form a pair ICThe affinity chromatography medium with fusion protein affinity has the structure of agarose microsphere-activated middle arm-IN *Affinity ligand:
(5)ICpurification of the expression of the fusion protein of the Gene of interest
Example 7
The target protein to be purified is connected to the C-terminal end of the C-terminal splicing domain of the NpuDnaE by using a fusion PCR technology. Using the methods of examples 2 and 3 above, the fusion DNA fragment was ligated to pET28a vector and transformed into competent E.coli BL21(DE3) to obtain ICAnd a fusion expression strain with a target protein GFP. Fusion of fragment I to the Gene of interest Using the conditions for inducible expression described in example 4C-expression of GFP.
Example 8
I of example 5CThe expression product of the fusion protein with the objective gene is expressed according to the method of example 4. The crude protein sample containing the fusion protein expression fragment was Buffer-exchanged with equilibrium Buffer A (1mmol/L Zn) by ultrafiltration2+(ii) a 20mmol/L imidazole; 0.5mol/L NaCl; 20mmol/L Na2HPO4；pH 6.5)。
Based on an AKTA purifier chromatography system, the specific process of purifying the target protein by utilizing the affinity chromatography system constructed by the invention is as follows:
1) and (4) balancing. 3 column volumes of equilibration Buffer A (1mmol/L Zn)2+(ii) a 20mmol/L imidazole; 0.5mol/L NaCl; 20mmol/LNa2HPO4(ii) a pH 6.5) the medium was equilibrated.
2) And (4) loading. The crude unpurified protein was loaded in solution, where the Buffer environment for the protein was Buffer A.
3) And (4) cleaning the hybrid protein. Unbound protein was removed by rinsing well with 4 column volumes of Buffer A. With 2 volumes free of Zn2+The Buffer A of (1) was washed.
4) Inducing fragmentation. 3 volumes of fragmentation Buffer B (50mmol/L DTT, 20mmol/L EDTA, 0.5mol/L NaCl, 10mmol LTris-HCl, pH 8.0) washes the chromatography medium and replaces the Buffer in the chromatography medium with fragmentation Buffer. Standing at room temperature for 3 h.
5) And (4) eluting. The cleaved target protein was washed with elution Buffer C (0.5mol/L NaCl, 10mmol/L Tris-HCl, pH 8.0) and collected.
(6)IN *Regeneration of affinity chromatography columns
Example 9
After the elution of the target protein was completed, Buffer D (0.5M NaCl, 50mmol/L Na) was added as a regeneration Buffer2HPO4NaOH, pH 11.4) binding to the affinity mediaCThe fractions were eluted, washed with 20% ethanol and stored at 4 ℃.
Repeating the purification of the target protein several times according to the above procedure, test IN *The purification efficiency of the affinity chromatographic column is not obviously reduced when the number of times of recycling is more than 40 times.
Packing the affinity chromatography medium into a column; with a Zn content of 1mmol/L2+The PBS buffer solution is used for balancing; will contain the protein of interest ICLoading the crude fusion protein sample; with no Zn content2+Washing with PBS buffer solution; washing the chromatography medium with a fragmentation buffer solution containing 50mmol/L DTT, and standing at room temperature for 3 h; eluting the fractured target protein by using an elution buffer solution and collecting; i isN *Regenerating the affinity chromatographic column.
As can be seen, I is expressed by the present inventionN *The affinity chromatography filler prepared by the affinity ligand has the following advantages:
(1) compared with the traditional affinity chromatography, the invention does not need any protease to cut the target protein, and is completed by the cleavage reaction of the intein, thereby reducing the cost and preventing the pollution to the target protein.
(2) Compared with some existing fragmentation intein purification systems, the affinity ligand fragment I is used in the inventionNThe C-terminal of (a) is linked to the medium while moving the position of His-tag to INThe N terminal of (1). By lowering INAnd ICThe steric hindrance in the process of mutual recognition and combination improves the fracture activity, and the fracture rate of 3h in the presence of a reducing agent reaches 95%.
(3) Zn realized by the invention2+Effectively control the fracture reaction and improve the purification efficiency of the system. However, the absence of mutation in the first amino acid results in an N-terminal cleavage reaction that cannot be prevented, which in turn leads to a splicing reaction. The splicing reaction of the intein can make the N-terminal extein sequence connect with the C-terminal extein sequence, i.e. adding the I-site at the N-terminal of the target protein GFPNAdditional amino acid sequence at the N-terminus of the fragment. In practical application of constructing intein affinity purification systemNThe fragments are covalently linked to the medium as affinity ligands and are used repeatedly, so that the phenomenon of adding amino acids to the N-terminus of the target protein occurs mostly in the first purification. Since the Asp118 mutation makes the C-terminal cleavage reaction independent of the N-terminus, it is theorized that I is the site ofNAfter all the amino acids at the N-terminal are transferred to the N-terminal of the target protein, the target protein GFP without additional amino acids can be generated. The fragmentation combination is carried out in a practical affinity purification systemFor commercial application, the product may be purified once to remove INThe additional amino acid at the N-terminus can then be used as a normal purification medium.
(4) In addition, the invention changes the traditional immobilization of I through affinity binding between Tag and mediumNFragment, replacing Tag with Cys and fixing affinity ligand I by covalent cross-linking of Cys and chromatographic microsphereN *. Experiments prove that the single target protein band is obtained by analyzing the purification result of the novel affinity purification medium-mediated purification system through SDS-PAGE, and the purification efficiency is 50.9 percent by protein concentration determination and calculation. The purification efficiency of the affinity purification medium is not obviously reduced after the affinity purification medium is repeatedly used for 40 times, which shows that the affinity purification medium constructed by the invention has better purification effect and higher repeated utilization rate.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. An affinity ligand characterized by: the affinity ligand was constructed on the surface of the NpuDnaE-N, i.e.INOn the basis of the modification of the structure of (1), modifiedNThe fragment is named as IN *(ii) a Wherein,
natural INThe C end of the chromatographic microsphere is added with cysteine for covalent binding with the chromatographic microsphere;
natural INThe first amino acid of the N end is introduced into mutation Cys1Ala, so that the occurrence of N end breakage reaction is avoided;
his-tag placed in nature INThe N-terminus of the fragment was used to purify the ligand fragment.
2. Affinity ligand according to claim 1, characterized in that: the natural NpuDnaE gene sequence template is shown in a sequence table SEQ ID No. 3.
3. Affinity ligand according to claim 2, characterized in that: the His-tag is placed in the natural INN-terminal of the fragment, mutant Cys1Ala, native INThe C end of the protein is added with cysteine, the gene sequence of the cysteine is shown as a sequence table SEQ ID No.4, and the protein sequence of the cysteine is shown as a sequence table SEQ ID No. 6.
4. A method for constructing an affinity ligand according to any one of claims 1 to 3, wherein: carrying out molecular modification on the N-terminal splicing structural domain of natural fractured intein NpuDnaE to obtain modified INThe fragment is named as IN *The method comprises the following steps of (1),
in nature INAdding a Cys to the C-terminal of (1);
in INAdding a His-tag sequence at the N end of the fragment;
adding to the primerNdeI. Hind III two cleavage sites and use them to cleave IN *The fragment is inserted into an expression vector pET28a, and the obtained protein is the affinity ligand fragment.
5. Method for the construction of affinity ligands according to claim 4, characterized in that: the primer, wherein, IN *The gene sequences of the upstream primer and the downstream primer with modified sequences are respectively shown as SEQ ID No.1 and SEQ ID No.2 of the sequence table.
6. Method for the construction of affinity ligands according to claim 4 or 5, characterized in that: also comprises the following steps of (1) preparing,
construction of recombinant bacteria, PCR is carried out by taking pRSFDuet-1-NpuDnaE plasmid as a template to obtain modified tabletsSegment IN *The recombinant plasmid pMD19-T-I is constructed by utilizing the vector pMD19-TN *Coli JM109 was transformed to be competent, and then the constructed I was cleaved with Nde I and Hind III enzymesN *The sequence is connected with expression plasmid pET-21b by enzyme digestion to obtain recombinant expression plasmid pET-21b-IN *Finally, competent E.coli BL21(DE3) was transformed to obtain the modified recombinant IN *Expression strain E.coli BL21(DE3)/pET-21b-IN *；
NpuDnaEIN *The recombinant bacterium E.coli BL21(DE3)/pET-21b-IN *Carrying out induced expression to obtain the modified recombinant IN *A protein.
7. Method for the construction of affinity ligands according to claim 6, characterized in that: and recombinant IN *Protein pairing ICThe gene sequence and the protein sequence of the GFP fragment are shown in the sequence tables SEQ ID No.5 and SEQ ID No. 7.
8. An affinity chromatography medium coupled with an affinity ligand according to any one of claims 1 to 3, wherein: with IN *The fragment is affinity ligand and is covalently coupled to agarose microspheres through activating a middle arm to form a pair ICThe affinity chromatography medium with fusion protein affinity has the structure of agarose microsphere-activated middle arm-IN *Affinity ligand:
9. a method for the coupled preparation of an affinity chromatography medium according to claim 8, wherein: the method comprises the following steps of,
activating agarose containing hydroxyl by using 1, 4-butanediol diglycidyl ether serving as a diepoxide to obtain activated epoxy agarose containing a long hydrophilic chain;
the ethylene oxide groups on the activated epoxy agarose and IN *The sulfhydryl of the fragment C-terminal Cys reacts to prepare the affinity ligand fragment IN *Coupling with activated agarose medium to obtain affinity chromatography medium.
10. Use of an affinity chromatography medium according to claim 8, wherein: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
packing the affinity chromatography medium into a column;
with a Zn content of 1mmol/L2+The PBS buffer solution is used for balancing;
will contain the protein of interest ICLoading the crude fusion protein sample;
with no Zn content2+Washing with PBS buffer solution;
washing the chromatography medium with a fragmentation buffer solution containing 50mmol/L DTT, and standing at room temperature for 3 h;
eluting the fractured target protein by using an elution buffer solution and collecting;
IN *regenerating the affinity chromatographic column.
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| TWI671314B (en) * | 2018-06-29 | 2019-09-11 | 國立清華大學 | Control self-cleavage protein activity and application thereof |
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