CN106543274B - Derivative of protein A structural domain Z with specific binding effect on antibody and application thereof - Google Patents
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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
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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- 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
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/282—Porous sorbents
- B01J20/285—Porous sorbents based on polymers
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- 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
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
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- 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- 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
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
- B01J2220/44—Materials comprising a mixture of organic materials
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Abstract
The invention relates to a derivative of a protein A structural domain Z with specific binding effect on an antibody and application thereof, wherein at least 1 amino acid residue except N23T mutation in an amino acid sequence of the structural domain Z is mutated. The derivative is obtained by a strategy of mutating at least one amino acid residue in Helix 1 and Helix 2 in protein A mutational structural domain Z. On the basis, the preparation method of the affinity chromatographic medium using the structural domain Z derivative as the ligand is provided. Compared with affinity chromatography media taking the structural domain Z and oligomers thereof as ligands, the structural domain Z derivative provided by the invention has higher chemical stability, and the protein denaturation temperature in an alkaline solution is increased by 4 ℃; the synthesized affinity chromatography medium maintains a comparable antibody adsorption capacity.
Description
Technical Field
The invention relates to a derivative of a protein A mutant structural domain Z with improved chemical stability under alkaline conditions and an application method thereof, in particular to a preparation method of an affinity chromatography medium taking the mutant structural domain Z derivative as a ligand, belonging to the field of affinity chromatography in biotechnology.
Background
Monoclonal antibody drug manufacturing technology has been rapidly developed over the last 30 years. This is mainly due to the construction of high expression cell lines, the improvement of culture media and the continuous scale-up of reactors. At present, a reactor with more than 20000 liters is popularized, the antibody titer is also increased by more than 30 times, and the antibody titer of 25g/L is also reported [ New Biotechnol,2011,28(5): 458-63; adv Drug Deliv Rev,2006,58(5-6):671-85 ]. Protein a chromatography is currently the most widely used and mature technique in antibody capture and purification, and is considered the core unit of antibody production and the "gold standard". Common commercial protein A chromatographic media mainly comprise media with natural protein A as ligand (such as protein A Sepharose Fast Flow of GE company) and media with recombinant protein A as ligand (such as rProtein A Sepharose Fast Flow of GE company and MabSelect SuRe). However, the currently used protein A chromatographic medium not only has limited processing capacity (20-30 g/L), but also has recognized defects of high cost, short service life, harsh operating conditions and the like. As a fundamental means to reduce the cost of chromatography media, reuse of protein a chromatography media is essential. Like protein chromatography techniques such as ion exchange, gel filtration, hydrophobic interaction, etc., protein a chromatography media after antibody recovery is cleaned by a well-established, standard clean-in-place (CIP) procedure to remove impurities such as contaminating proteins, residual antibodies and their polymers, endotoxins, etc. from the media. A standard protein chromatography CIP procedure involves treating the chromatographic media with 0.5mol/L to 1.0mol/L sodium hydroxide solution. Such conditions are very harsh for protein a chromatography media, with the direct consequences of disruption of the protein a higher structure, reduction of the adsorption capacity of the media and reduction of the lifetime. Therefore, protein A chromatography has become the main technical "bottleneck" of monoclonal antibody drug preparation and the key engineering technology to be broken through urgently.
Protein a is a 42kDa molecular weight protein molecule isolated from the cell wall of Staphylococcus aureus (Staphylococcus aureus) that contains highly homologous antibody binding domains E, D, A, B and C, each consisting of about 58 amino acid residues. The five antibody domains have more advantages in terms of the binding specificity of the domain B to the antibody. On the basis, the mutation structural domain Z obtained by mutating 29 th glycine on the structural domain B into alanine (G29A) not only eliminates the chemical dissociation of the structural domain B caused by deamidation, but also improves the stability of the structural domain B under the alkaline condition [ Chinese patent publication No. CN101522278A ]. In addition, the binding effect of the domain to the Fab fragment of the antibody is also reduced with mutation, and the binding specificity to the Fc fragment of the antibody is enhanced [ chinese patent application No.: 201080013514.0 ]. In view of better alkali resistance, domain Z and oligomers thereof have been widely used in protein a chromatography media. The resistance of the domain Z to alkaline lye is further increased by the N23T mutation [ Proteins-Structure Function and Bioinformatics,2004,55(2): 407-. The publication CN1642976A further discloses a method for preparing immunoglobulin-binding proteins in which at least one asparagine residue is mutated, characterized in that the mutations comprise different combinations of N23T and N43E, N28A, N6A, N11S, N11S and N23T, and N6A and N23T. This strategy is reflected only in the engineering of a specific amino acid (asparagine). Minakuchi et al reported that the G29A mutation in antibody domain C also improved the alkali resistance of domain C [ Protein Science,2013,22(9):1230-1238 ]. In contrast, the antibody domain C has a G29Y, G29M or G29W mutation with more advantageous alkali resistance. Bell chemical company discloses a mutant of antibody domains E, D, A, B and C obtained by mutating glycine to an amino acid other than alanine [ publication No. CN 102365361A ], which is characterized in that the affinity of the obtained mutant domain to an antibody Fab fragment is reduced and the chemical stability under alkaline conditions is improved. These methods of improving the alkali resistance of antibody domains have been developed for deamidation and specific amino acids.
Currently, protein a chromatography ligands, represented by domain Z and oligomers thereof, have been widely used in the purification of antibodies. The chromatographic process described above comprises a CIP step using a 0.1mol/L sodium hydroxide solution to remove residual proteins and impurities from the chromatographic medium. This is in contrast to the 0.5-1.0 mol/L sodium hydroxide solution required by the standard CIP [ publication No.: CN101522278A ]. The strategy of standard CIP is to expose the chromatographic medium to an environment with a pH above 13. This is still very harsh on the currently commercially available Protein A chromatography media, which leads to a disruption or even loss of the steric structure of the Protein A chromatography ligand and a reduction in the binding capacity of the antibody [ Protein Science,2013,22(9):1230-1238 ]. Therefore, further improving the alkali resistance of domain Z is key to solving the safety and cost of current protein a chromatography application.
In addition to eliminating deamidation of antibody domains, attempts to address protein ligand binding specificity and chemical stability have also been made from other perspectives. Deisenhofer identified 11 key amino acid residues involved in binding in antibody domain B by analyzing the crystal structure of the complex of the antibody Fc fragment and protein A antibody domain B [ Biochemistry,1981,20:2361-70 ]. Palmer et al reported that a Y3F/T16I/T18I mutant of the antibody-binding protein G molecule isolated from the cell wall of Streptococcus has the feature of better alkali resistance than the wild-type protein G [ Journal of Biotechnology,2008,134(3-4):222-230 ]. Lionhexi and lisishu disclose a novel method for obtaining low antibody affinity protein a, characterized by the double F13G/Y14G mutation in domain Z [ publication No.: CN 105198973a ]. The antibody affinity of the mutant is reduced by more than 90% compared with the structural domain Z. In order to systematically improve the binding specificity and chemical stability of the domain Z or one of the two, an overall strategy for stabilizing the domain Z is provided on the basis of the deep knowledge of the stability of the domain Z and the molecular mechanism of the interaction of the domain Z with an antibody, and a domain Z derivative with better chemical stability is obtained by stabilizing the overall structure of the domain Z.
Disclosure of Invention
The invention aims to improve the chemical stability of the structural domain Z in alkaline solution, thereby providing a derivative of the structural domain Z with specific binding action on an antibody, which has good chemical stability under alkaline conditions; on the basis, the preparation method of the affinity chromatographic medium using the structural domain Z derivative as the ligand is provided. Compared with affinity chromatographic medium using structure domain Z and oligomer thereof as ligand, the affinity chromatographic medium prepared by the invention has equivalent antibody binding capacity.
The object of the present invention is achieved by the following technical means.
A derivative of domain Z of protein A having specific binding to an antibody, characterized in that said derivative of domain Z is mutated in the amino acid sequence of domain Z from SEQ ID No. 1 by at least 1 amino acid residue in addition to the N23T mutation, said derivative of domain Z having acquired a higher chemical stability under alkaline conditions than the parent molecule.
The derivative having a specific binding effect to an antibody is characterized in that the domain Z derivative can be specifically bound with an Fc fragment of the antibody.
The domain Z derivative is characterized in that the parent molecule comprises a sequence of SEQ ID No. 1 and the mutation of the amino acid is selected from at least 1 amino acid residue of 23-position asparagine residue and 11-position asparagine residue, 12-position alanine residue, 32-position glutamine residue and 35-position lysine residue.
The domain Z derivative is characterized in that asparagine at position 11, alanine at position 12, glutamine at position 32 and lysine at position 35 are mutated, and more preferably, asparagine residue at position 11 is mutated to threonine, alanine residue at position 12 is mutated to tryptophan, glutamine residue at position 32 is mutated to threonine, and lysine residue at position 35 is mutated to arginine. .
A multidomain protein comprising 2 or more of said domain Z derivatives linked together, wherein the number of domain Z derivatives is from 2 to 6.
The multidomain protein is characterized in that each domain is formed by connecting a plurality of glycine residues.
The method for preparing the affinity chromatographic medium by using the mutant domain Z derivative as the ligand is characterized in that the domain Z derivative or the derivative polymer with the average grain diameter of 30-300 mu m is coupled in an agarose gel particle pore channel.
The affinity chromatographic medium is characterized in that the density of the domain Z derivative or derivative polymer ligand of the affinity chromatographic medium is 2.0-15 mg/mL.
The affinity chromatography medium is characterized in that the affinity chromatography medium can be combined with protein containing Fc fragments of antibodies.
The method for affinity chromatography media for antibody separation, wherein the domain Z derivative or derivative multimer is coupled to the sepharose particles via an amino group or a carboxyl group.
Compared with parent molecules, the derivative of the domain Z provided by the invention has better chemical stability under alkaline conditions, and the binding specificity of the antibody of the derivative is equivalent to that of the domain Z. Affinity chromatography media prepared on the basis and using the structural domain Z derivative as the ligand can tolerate CIP cleaning of lye with higher concentration and maintain equivalent antibody binding capacity compared with affinity chromatography media using parent molecules as the ligand.
Compared with the prior art, the structural domain Z derivative and the affinity chromatographic medium taking the structural domain Z derivative as the ligand have the following advantages:
(1) the domain Z derivatives provided by the present invention are based on the overall stabilization strategy of the protein structure. Implementation of this strategy was based on a profound understanding of the structure of the domain Z protein. The improvement of the chemical stability of the derivatives of the structural domain Z is not limited to the mutation of a single or only certain amino acid residue in the sequence (such as G29A or the mutation of asparagine residue and the like), but is derived from the mutation of at least one amino acid residue in Helix 1 and Helix 2 in the structural domain Z, and shows an overall strategy for stabilizing the molecular structure of the protein.
(2) The domain Z derivative provided by the invention has higher chemical stability. Compared with the structural domain Z of SEQ ID No. 1, the protein denaturation temperature of the derivative with the sequence number of SEQ ID No. 2 in 0.1mol/L sodium hydroxide solution is increased by 4 ℃, and the protein denaturation temperature of the derivative with the sequence number of SEQ ID No. 3 is increased by 3.2 ℃. This result shows that the domain Z derivative provided by the present invention has higher stability.
(3) Affinity chromatography media based on domain Z derivatives as ligands maintain comparable antibody binding capacity. The domain Z derivative provided by the invention is obtained on the basis of the integral stability strategy of the protein molecular structure, and the mutation of the amino acid residue does not relate to the key amino acid residue with the binding effect with the Fc fragment of the antibody or does not change the property of the key amino acid residue with the binding effect with the Fc fragment of the antibody. Thus, the obtained domain Z derivative substantially maintains comparable antibody binding capacity and adsorption capacity.
Drawings
In order that the present invention may be more readily and clearly understood, reference is now made to the following detailed description taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a differential scanning calorimetry result of protein A domain Z of SEQ ID No. 1-4 and its derivatives in a 20mmol/L phosphate buffer containing 100mmol/L sodium chloride and 0.1mol/L sodium hydroxide solution.
FIG. 2 shows the recovery of the domain Z derivative expression products of SEQ ID Nos. 5(a) and 7(b) by ion exchange chromatography on Q Sepharose FF.
FIG. 3 shows the results of antibody adsorption of affinity chromatography media using a domain Z derivative as a ligand in a 20mmol/L phosphate buffer containing 100mmol/L sodium chloride.
Detailed Description
The present embodiments are provided for illustrative purposes only and should not be construed to limit the present invention as defined in the appended claims.
Example 1 preparation and stability of Domain Z derivative of SEQ ID No. 2
And respectively synthesizing the structural domain Z with the sequence number of SEQ ID No. 1 and the protein with the sequence number of SEQ ID No. 2 by using a polypeptide solid phase chemical synthesis method. The protein was dissolved in 20mmol/L PBS buffer (pH 6.0) containing 100mmol/L NaCl, and the resulting solution was prepared as a protein solution having a protein concentration of 1 mg/mL. The denaturation temperatures of the proteins in the two solutions mentioned above were carried out in a differential scanning calorimeter of the VP-DSC type. First, after the sample cell and the reference cell of VP-DSC were washed with 100mL of ultrapure water, about 0.75mL each of a buffer solution (20 mmol/L PBS buffer solution containing 100mmol/L NaCl, pH 6.0) and a protein solution degassed under vacuum for 15min or more was added to the reference cell and the sample cell, respectively, using a Hamilton syringe (the volumes of the reference cell and the sample cell were 0.5282 mL). After the excess solution was removed by the Hamilton sample feeder, the sample and reference cells were capped and sealed. The temperature scanning range of differential scanning calorimetry is 25-120 deg.C, and the heating rate is 1 deg.C/min. The test temperature equilibration time was 8min before heating began. The data processing is completed by MicroCal-enabled Origin 7.0 software. The results are shown in FIG. 1. The results showed that the denaturation temperature of domain Z of SEQ ID No. 1 was 75.3 ℃ and the denaturation temperature of the domain Z derivative of SEQ ID No. 2 was 79.3 ℃. This indicates that the domain Z derivative of SEQ ID No. 2 is more stable under neutral conditions.
Example 2 stability of Domain Z derivative of SEQ ID No. 2 in alkaline solution
The domain Z of SEQ ID No. 1 and the protein of SEQ ID No. 2 were synthesized using the preparation method described in example 1. The protein was dissolved in 0.1mol/L sodium hydroxide solution to prepare a protein solution having a protein concentration of 1 mg/mL. The denaturation temperatures of the proteins in the above two solutions were analyzed by differential scanning calorimetry as in example 1. The solution system is 0.1mol/L sodium hydroxide solution. The reference cell was filled with 0.1mol/L NaOH solution degassed under vacuum for more than 15 min. The temperature scanning range of differential scanning calorimetry is 25-120 deg.C, and the heating rate is 1 deg.C/min. The test temperature equilibration time was 8min before heating began. The data processing is completed by MicroCal-enabled Origin 7.0 software. The results are shown in FIG. 1. The results showed that the denaturation temperature of the domain Z of SEQ ID No. 1 in 0.1mol/L NaOH solution was 61.6 ℃ and the denaturation temperature of the domain Z derivative of SEQ ID No. 2 in 0.1mol/L NaOH solution was 64.8 ℃. This indicates that the domain Z derivative of SEQ ID No. 2 is more chemically stable in 0.1mol/L NaOH solution.
Example 3 preparation and stability of Domain Z derivative of SEQ ID No. 3
The protein with the sequence number of SEQ ID No. 3 was synthesized using the preparation method described in example 1. The denaturation temperature of the above-mentioned proteins under neutral conditions was analyzed by the differential scanning calorimetry as in example 1. The results are shown in FIG. 1. The results showed that the denaturation temperature of the domain Z derivative of SEQ ID No. 3 was 76.9 ℃. This indicates that the domain Z derivative of sequence No. SEQID No. 3 is more stable under neutral conditions than domain Z.
Example 4 stability of Domain Z derivative of SEQ ID No. 3 in alkaline solution
The stability of the domain Z derivative with the sequence number SEQ ID No. 3 in alkaline solution was measured using the method described in example 2. The results are shown in FIG. 1. The results showed that the denaturation temperature of the domain Z derivative of SEQ ID No. 3 in 0.1mol/L NaOH solution was 62.8 ℃. This indicates that the domain Z derivative of SEQ ID No. 3 is more chemically stable in 0.1mol/L NaOH solution.
Example 5 preparation of Domain Z derivative of SEQ ID No. 4 and stability measurement under various conditions
The protein with the sequence number of SEQ ID No. 4 was synthesized using the preparation method described in example 1. The denaturation temperature of the above proteins under neutral and alkaline conditions was analyzed by differential scanning calorimetry as in examples 1 and 2. The results are shown in FIG. 1. The results showed that the domain Z derivative of SEQ ID No. 4 had a denaturation temperature of 77.5 ℃ under neutral conditions and a denaturation temperature of 62.4 ℃ in 0.1mol/L NaOH solution. This indicates that the domain Z derivative of SEQ ID No. 4 is superior to the domain Z in chemical stability under neutral conditions and in 0.1mol/L NaOH solution.
Example 6 preparation of Domain Z derivative having sequence No. SEQ ID No. 5
A structural domain Z derivative gene sequence with the sequence number of SEQ ID No. 5 is designed and synthesized by a chemical synthesis method, and the length of the sequence of SEQ ID No. 6 is 375 bp. The above sequence includes the stop codon TAA as well as the added 5 'Ndel and 3' Xhol. 5 'Ndel and 3' Xhol were used for cloning into the vector pet-30a (Kan). The vector is cut by restriction enzymes Ndel and Xhol and then is connected with the gene SEQ ID No. 5 to obtain pet-30a (Kan) plasmid containing the gene SEQ ID No. 6. The plasmid was transformed into BL21DE3 cells by the calcium chloride method. Selecting single colony of BL21DE3 transformed Escherichia coli, inoculating into liquid LB culture medium containing 30 μ g/mL kanamycin, culturing overnight, inoculating into fermentation broth under aseptic condition with inoculation concentration of not less than 1:200(v/v), fermenting at 37 deg.C until OD is not less than OD600When the value reaches 0.6-0.8, adding IPTG with the concentration of 500mmol/L until the final concentration of IPTG in the fermentation liquor is 0.5mmol/L, and continuing to ferment for more than 6 hours. The collected fermentation broth was centrifuged at 4800rpm for 30min to remove the supernatant, and then the cells were resuspended in 20mmol/L Tris-HCl buffer (pH 7.5). After repeating the above operation, the suspension of the cells was sonicated and the disruption solution was collected. Filtering the cell disruption solution with 0.45 μm filter membrane, and performing Q-Sepharose HP ion exchange chromatographyPurification was carried out using 20mmol/L Tris-HCl buffer (pH7.5) as the adsorption buffer and 20mmol/L Tris-HCl buffer (pH7.5) containing 1.0mol/L NaCl as the elution buffer. And recovering the target protein by gradient elution under the condition that the elution buffer solution is linearly increased to 15 percent within 30 min. The chromatographic peak eluting at a conductivity of 8-11mS/cm was collected. The peak results of the chromatography are shown in FIG. 2 (a). The purity of the target protein was 96%.
Example 7 preparation of Domain Z derivative having sequence No. SEQ ID No. 7
By adopting the method described in the embodiment 6, the structural domain Z derivative gene sequence with the sequence number of SEQ ID No. 7 is designed and synthesized by a chemical synthesis method, and the length of the sequence of SEQ ID No. 8 is 747 bp. The vector is cut by restriction enzymes Ndel and Xhol and then is connected with the gene SEQ ID No. 8 to obtain pet-30a (Kan) plasmid containing the gene SEQ ID No. 8. The plasmid was transformed into BL21DE3 cells by the calcium chloride method. The chromatographic peak results after microbial fermentation and separation and purification are shown in FIG. 2 (b). The purity of the target protein obtained was 95.2%.
Example 8 preparation of affinity chromatography media based on ligand with Domain Z derivative of SEQ ID No. 2
Affinity chromatography media were prepared using the domain Z derivative of sequence No. SEQ ID No. 2 synthesized in example 1 as ligand. The preparation process comprises two steps of medium activation and ligand coupling. The medium activation method is as follows: after Sepharose FF stored in 20% ethanol is rinsed with copious amounts of deionized water and drained from a G3 funnel, 15.0G of the drained media are weighed into a 100mL Erlenmeyer flask, 7.5mL of 75% (w/w) glycidyltrimethylammonium chloride (GTMAC), 7.5mL of double distilled water and 0.387G of 50% (w/w) sodium hydroxide solution are added, mixed well, and left to react overnight at 25 ℃ at 170 rpm; after the reaction is finished, cleaning the medium by a large amount of deionized water and carrying out pumping treatment, weighing 13.0g of pumping medium, transferring the medium into a 100mL triangular flask, and adding 8.0mL of 4.6mol/L NaOH solution for full suspension; then, 11.0mL of 1, 4-butanediol diglycidyl ether (BDDE) was added and reacted at 37 ℃ and 170rpm for 2 hours; after the reaction is finished, washing the reaction product by using a large amount of deionized water to obtain the activated chromatographic medium with the epoxy group density of 38 mu mol/g. The ligand coupling method is as follows: weighing 4.0g of activation medium, placing the activation medium in a 50mL conical flask, adding 10mmol/L sodium bicarbonate solution and 20mL for balancing for 1h, draining the solution, transferring the medium to a 25mL conical flask, adding 8.5mL of 10mmol/L sodium bicarbonate solution containing 40mg of domain Z derivative with the sequence number of SEQ ID No. 2, fully suspending, and reacting for 2h at 37 ℃ and 170 rpm; after the reaction is finished, cleaning the medium by using a large amount of deionized water and performing pumping treatment; the chromatographic medium obtained was placed in a 25mL Erlenmeyer flask, 8mL blocking buffer was added and the reaction was carried out overnight at 25 ℃ and 170 rpm; the reaction product was washed three times with 0.1mol/L Tris-HCl buffer (pH 8.0) containing 0.15mol/L NaCl and 50mmol/L acetate buffer (pH 4.5) in this order, and the washed media were finally washed with deionized water and 20% ethanol, respectively, to obtain affinity chromatography medium Z2Sepahrose FF with ligand density of 6.3 mg/g.
Adding 8.5mL of 10mmol/L sodium bicarbonate solution containing 40mg of the structural domain Z with the sequence number of SEQ ID No. 1 by adopting the synthesis method, fully suspending, and reacting for 2h at 37 ℃ and 170 rpm; the affinity chromatography medium Z1Sepharose FF with a ligand density of 5.7mg/g was obtained.
The antibody adsorption equilibrium curves of the above affinity chromatography media Z2Sepharose FF and Z1Sepharose FF in a 20mmol/L phosphate buffer solution (pH7.4) containing 100mmol/L sodium chloride are shown in FIG. 3, and the saturated adsorption capacities of the corresponding antibodies are 69.0mg/g medium and 60.9mg/g medium, respectively.
Example 9 preparation of affinity chromatography media based on ligand with Domain Z derivative of SEQ ID No. 3
An affinity chromatography medium using the domain Z derivative of SEQ ID No. 3 as ligand was prepared as described in example 8, wherein 4g of the activation medium was added to 8.0mL of a 10mmol/L sodium bicarbonate solution containing 40mg of the domain Z derivative having the sequence number of SEQ ID No. 3, and after sufficient suspension, the reaction was carried out at 37 ℃ and 170rpm to obtain an affinity chromatography medium Z3Sepharose FF having a ligand density of 7.1 mg/g. FIG. 3 shows the adsorption equilibrium curve of the antibody on the affinity chromatography medium in a 20mmol/L phosphate buffer solution (pH7.4) containing 100mmol/L sodium chloride, and the saturation adsorption capacity of the antibody is 52.3mg/g medium.
Example 10 preparation of affinity chromatography media based on ligand with Domain Z derivative of SEQ ID No. 4
An affinity chromatography medium using the domain Z derivative of SEQ ID No. 4 as ligand was prepared as described in example 8, wherein 8.0mL of 10mmol/L sodium bicarbonate solution containing 100mg of the domain Z derivative of SEQ ID No. 4 was added to 4g of the activation medium, and after sufficient suspension, the reaction was carried out at 37 ℃ and 170rpm to obtain an affinity chromatography medium Z4Sepharose FF with a ligand density of 14.8 mg/g. The adsorption equilibrium curve of the antibody on the affinity chromatography medium in a 20mmol/L phosphate buffer solution (pH7.4) containing 100mmol/L sodium chloride is shown in FIG. 3, and the saturation adsorption capacity of the antibody is 45.8mg/g medium.
Example 11 preparation of affinity chromatography media based on ligand with Domain Z derivative of SEQ ID No. 5
An affinity chromatography medium using the domain Z derivative of SEQ ID No. 5 as ligand was prepared as described in example 8, wherein 75% (w/w) glycidyltrimethylammonium chloride (GTMAC)20.0mL and 1, 4-butanediol diglycidyl ether (BDDE) 20.0mL were added during activation of the medium, and the reaction yielded an activated chromatography medium with an epoxy density of 77.1. mu. mol/g. During ligand coupling, 8.5mL of 10mmol/L sodium bicarbonate solution containing 20mg of the domain Z derivative having the sequence number of SEQ ID No. 5 prepared according to example 5 was added to 4.0g of activation medium, and reacted to obtain affinity chromatography medium Z5Sepahrose FF having a ligand density of 2.3 mg/g. FIG. 3 shows the antibody adsorption equilibrium curve of the affinity chromatography medium in 20mmol/L phosphate buffer (pH7.4) containing 100mmol/L sodium chloride, and the saturation adsorption capacity of the antibody is 31.1mg/g medium.
Example 12 preparation of affinity chromatography media based on ligand with Domain Z derivative of SEQ ID No. 7
An affinity chromatography medium using the domain Z derivative of SEQ ID No. 7 as ligand was prepared as described in example 9, wherein 8.5mL of 10mmol/L sodium bicarbonate solution containing 30mg of the domain Z derivative of SEQ ID No. 7 prepared according to example 7 was added to 4.0g of activation medium during ligand coupling, and the reaction yielded an affinity chromatography medium Z7Sepahrose FF with a ligand density of 6.6 mg/g. The adsorption equilibrium curve of the antibody on the affinity chromatography medium in 20mmol/L phosphate buffer (pH7.4) containing 100mmol/L sodium chloride is shown in FIG. 3, and the saturation adsorption capacity of the antibody is 72.7mg/g medium.
The invention discloses and proposes a staphylococcal protein A domain Z derivative with improved stability and application thereof, which can be realized by those skilled in the art by referring to the contents in the text, and appropriately changing the conditional routes and the like, although the method and the preparation technology of the invention have been described by preferred embodiments, the related technical personnel can obviously modify or recombine the method and the technical routes described in the text to realize the final preparation technology without departing from the contents, the spirit and the scope of the invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and content of the invention.
SEQ ID No.:1
SEQ ID No.:2
SEQ ID No.:3
SEQ ID No.:4
SEQ ID No.:5
SEQ ID No.:6
CATATGGTGGATAACAAGTTCAATAAAGAGCAGCAGACCATTTTTTATGAAATTCTGCATCTGCCGAATCTGACCGAAGAACAGCGCAACGCCTTCATCACCAGTCTGCGTGATGATCCGAGCCAGAGCGCCAATCTGCTGGCCGAAGCCAAAAAGCTGAACGATGCCCAGGCACCGAAGG GCGGTGGCGGTG TGGACAATAAGTTTAACAAAGAGCAACAAACCATCTTCTACGAAATCCTGCACCTGCCGAACCTGACCGAGGAACAACGCAATGCCTTCATTACCAGCCTGCGTGATGACCCGAGCCAAAGTGCCAACCTGCTGGCCGAAGCCAAGAAGCTGAATGATGCCCAAGCCCCGAAATAACTC GAG
SEQ ID No.:7
SEQ ID No.:8
CATATGGTGGATAACAAGTTCAATAAAGAGCAGCAGACCATTTTTTATGAAATTCTGCATCTGCCGAATCTGACCGAAGAACAGCGCAACGCCTTCATCACCAGTCTGCGTGATGATCCGAGCCAGAGCGCCAATCTGCTGGCCGAAGCCAAAAAGCTGAACGATGCCCAGGCACCGAAGGGCGGTGGCGGTGTGGACAATAAGTTTAACAAAGAGCAACAAACCATCTTCTACGAAATCCTGCACCTGCCGAACCTGACCGAGGAACAACGCAATGCCTTCATTACCAGCCTGCGTGATGACCCGAGCCAAAGTGCCAACCTGCTGGCCGAAGCCAAGAAGCTGAATGATGCCCAAGCCCCGAAAGGTGGCGGTGGTGTTGATAATAAATTTAATAAGGAACAACAGACCATCTTTTATGAAATCCTGCATCTGCCGAACCTGACAGAGGAGCAGCGCAACGCCTTTATCACCAGTCTGCGCGACGATCCGAGCCAAAGCGCCAATCTGCTGGCCGAGGCCAAGAAACTGAATGATGCACAGGCACCTAAAGGCGGTGGCGGCGTGGACAACAAATTTAACAAGGAACAGCAGACCATCTTCTATGAGATCCTGCACCTGCCGAATCTGACCGAAGAACAGCGCAATGCCTTTATTACCAGCCTGCGCGATGATCCTAGCCAGAGTGCCAACCTGCTGGCCGAAGCAAAAAAGCTGAATGACGCCCAGGCCCCGAAGTAACTCGAG
Claims (6)
1. A protein A structural domain Z derivative with specific binding effect on an antibody is characterized in that the structural domain Z derivative is SEQ ID No. 2.
2. A multidomain protein comprising a derivative of protein A Domain Z having a specific binding activity for an antibody, wherein said multidomain protein comprises 2 or more domain Z derivatives linked together, the number of domain Z derivatives being from 2 to 6, and said domain Z derivative being SEQ ID NO. 2.
3. Multidomain protein of a derivative of protein a domain Z according to claim 2, characterised in that said multidomain protein is formed by several glycine residues connecting the individual domains.
4. An affinity chromatography medium comprising as ligand a derivative of domain Z of protein a of claim 1 wherein the affinity chromatography medium has a domain Z derivative or derivative multimer ligand density of 2.0-15 mg/mL.
5. The affinity chromatography media of claim 4, wherein the affinity chromatography media binds to a protein comprising an Fc fragment of an antibody.
6. The affinity chromatography media according to claim 4, wherein the domain Z derivative or derivative multimer is coupled to the sepharose particles via an amino or carboxyl group.
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|---|---|---|---|---|
| CN101087637A (en) * | 2004-12-14 | 2007-12-12 | 通用电气健康护理生物科学股份公司 | Purification of immunoglobulins |
| CN102272145A (en) * | 2009-01-12 | 2011-12-07 | 通用电气健康护理生物科学股份公司 | affinity chromatography matrix |
| CN102558318A (en) * | 2002-03-25 | 2012-07-11 | 通用电气健康护理生物科学股份公司 | A mutated immunoglobulin-binding protein |
| CN103269762A (en) * | 2010-12-20 | 2013-08-28 | 通用电气健康护理生物科学股份公司 | Affinity chromatography matrix |
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| CN102558318A (en) * | 2002-03-25 | 2012-07-11 | 通用电气健康护理生物科学股份公司 | A mutated immunoglobulin-binding protein |
| CN101087637A (en) * | 2004-12-14 | 2007-12-12 | 通用电气健康护理生物科学股份公司 | Purification of immunoglobulins |
| CN102272145A (en) * | 2009-01-12 | 2011-12-07 | 通用电气健康护理生物科学股份公司 | affinity chromatography matrix |
| CN103269762A (en) * | 2010-12-20 | 2013-08-28 | 通用电气健康护理生物科学股份公司 | Affinity chromatography matrix |
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