CN106543274B - A kind of derivative of protein A domain Z with specific binding effect on antibody and application thereof - Google Patents
A kind of derivative of protein A domain Z with specific binding effect on antibody and application thereof Download PDFInfo
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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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- 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
- B01J20/291—Gel sorbents
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- 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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- 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
本发明涉及一种对抗体具有特异性结合作用的A蛋白结构域Z的衍生物及其应用,结构域Z氨基酸序列中除N23T突变以外还有至少1个氨基酸残基发生突变。该衍生物通过A蛋白突变结构域Z中Helix 1和2中至少一个氨基酸残基突变的策略而获得。在此基础上,提供利用该结构域Z衍生物为配基的亲和色谱介质制备方法。与以结构域Z及其寡聚体为配基的亲和色谱介质相比,本发明提供的结构域Z衍生物具有更高的化学稳定性,在碱性溶液中的蛋白变性温度提高了4℃;所合成的亲和色谱介质维持了相当的抗体吸附容量。
The present invention relates to a protein A domain Z derivative with specific binding effect on antibodies and its application. In addition to the N23T mutation, at least one amino acid residue in the domain Z amino acid sequence is mutated. This derivative was obtained by a strategy of mutating at least one amino acid residue in Helix 1 and 2 in protein A mutation domain Z. On this basis, a method for preparing an affinity chromatography medium using the domain Z derivative as a ligand is provided. Compared with the affinity chromatography medium with domain Z and its oligomers as ligands, the domain Z derivatives provided by the present invention have higher chemical stability, and the protein denaturation temperature in alkaline solution is increased by 4 ℃; the synthesized affinity chromatography medium maintained a comparable antibody adsorption capacity.
Description
技术领域technical field
本发明涉及一种在碱性条件下具有改良化学稳定性的A蛋白突变结构域Z的衍生物及其应用的方法,具体是一种以突变结构域Z衍生物为配基的亲和色谱介质制备方法,属于生物技术中的亲和色谱领域。The present invention relates to a derivative of protein A mutant domain Z with improved chemical stability under alkaline conditions and a method for its application, in particular to an affinity chromatography medium using the mutant domain Z derivative as ligand The preparation method belongs to the field of affinity chromatography in biotechnology.
背景技术Background technique
单克隆抗体药物制备技术在过去的30年间获得了迅猛发展。这主要得益于高表达细胞系的构建、培养基的改良以及反应器规模不断扩大。目前,20000升以上的反应器已经开始推广,抗体滴度也增加了30多倍且25g/L的抗体滴度亦见报道【New Biotechnol,2011,28(5):458-63;Adv Drug Deliv Rev,2006,58(5-6):671-85】。A蛋白色谱则是目前抗体捕集和纯化过程中使用最为广泛和成熟的技术,其被视为抗体制备的核心单元和“金标准”。常见的商品化A蛋白色谱介质主要包括天然A蛋白为配基的介质(如GE公司的nProtein ASepharose Fast Flow等)和重组A蛋白为配基的介质(如GE公司的rProtein A SepharoseFast Flow和MabSelect SuRe等)。但是当前常用A蛋白色谱介质不仅处理能力有限(20-30g/L),而且存在成本高、寿命短和操作条件苛刻等公认的缺陷。作为降低色谱介质成本的基本手段,A蛋白色谱介质的重复使用是必需的。如同离子交换、凝胶过滤、疏水性相互作用等蛋白质色谱技术,回收抗体后A蛋白色谱介质需经严格验证的、标准的原位清洗(CIP)流程清洁以除去介质上的杂蛋白、残留抗体及其聚合物、内毒素等杂质。一个标准的蛋白质色谱CIP流程包括利用0.5mol/L至1.0mol/L氢氧化钠溶液处理色谱介质。这种条件对于A蛋白色谱介质而言是非常苛刻的,其直接结果就是A蛋白高级结构的破坏、介质吸附能力的下降和寿命的降低。因此,A蛋白色谱已成为单抗药物制备的主要技术“瓶颈”和亟待突破的重点工程技术。Monoclonal antibody drug preparation technology has developed rapidly in the past 30 years. This is mainly due to the construction of high-expressing cell lines, the improvement of the medium and the continuous expansion of the reactor scale. At present, reactors of more than 20,000 liters have been promoted, and the antibody titer has increased by more than 30 times, and the antibody titer of 25g/L has also been reported [New Biotechnol, 2011, 28(5): 458-63; Adv Drug Deliv Rev, 2006, 58(5-6):671-85]. Protein A chromatography is the most widely used and mature technology in the process of antibody capture and purification, and it is regarded as the core unit and "gold standard" of antibody preparation. Common commercial protein A chromatography media mainly include natural protein A as ligand media (such as GE's nProtein ASepharose Fast Flow, etc.) and recombinant protein A as ligand media (such as GE's rProtein A SepharoseFast Flow and MabSelect SuRe). Wait). However, currently commonly used protein A chromatography media not only have limited processing capacity (20-30 g/L), but also have recognized defects such as high cost, short life and harsh operating conditions. As a basic means to reduce the cost of chromatography media, the reuse of protein A chromatography media is necessary. Like ion exchange, gel filtration, hydrophobic interaction and other protein chromatography techniques, the protein A chromatography medium after recovery of antibodies needs to be cleaned by a rigorously validated and standard cleaning-in-place (CIP) process to remove impurities and residual antibodies on the medium. And its polymers, endotoxins and other impurities. A standard CIP procedure for protein chromatography involves treating the chromatography medium with 0.5 mol/L to 1.0 mol/L sodium hydroxide solution. This condition is very harsh for protein A chromatography media, and the direct result is the destruction of protein A's higher-order structure, the decline of the adsorption capacity of the media and the reduction of lifespan. Therefore, protein A chromatography has become the main technical "bottleneck" for the preparation of monoclonal antibodies and a key engineering technology that needs to be broken through.
A蛋白是从金黄色葡萄球菌(Staphylococcus aureus)细胞壁分离得到的一种分子量为42kDa的蛋白质分子,其包含各由约58个氨基酸残基构成且高度同源的抗体结合域E、D、A、B和C。上述五个抗体结构域中尤以结构域B与抗体结合特异性更具优势。在此基础上,通过对结构域B上29位甘氨酸突变为丙氨酸(G29A)获得的突变结构域Z不仅消除了脱酰胺作用引起的结构域B化学解离,提高了其在碱性条件下的稳定性【中国发明专利公开号CN101522278A】。此外,结构域与抗体Fab片段的结合作用也随着突变而降低,进而与抗体Fc片段的结合特异性增强【中国发明专利,申请号:201080013514.0】。有鉴于更好的耐碱性,结构域Z及其寡聚体已经被广泛地应用于A蛋白色谱介质。结构域Z对碱液的耐受性通过N23T突变获得了进一步的提升【Proteins-Structure Function and Bioinformatics,2004,55(2):407-416】。公开号为CN1642976A的专利进一步公开了至少一个天冬酰胺残基被突变的免疫球蛋白结合蛋白的方法,其特征在于突变包括了N23T和N43E、N28A、N6A、N11S、N11S和N23T以及N6A和N23T等不同组合。但这一策略仅反映在对于特定氨基酸(天冬酰胺)的改造。Minakuchi等人报道了抗体结构域C的G29A突变同样可以改善结构域C的耐碱性【Protein Science,2013,22(9):1230-1238】。与此相对应,抗体结构域C中G29Y、G29M或G29W突变的耐碱性更具优势。钟化株式会社则公开了一种将甘氨酸突变为除丙氨酸以外氨基酸的抗体结构域E、D、A、B和C的突变体【公布号CN 102365361A】,其特征在于获得的突变结构域与抗体Fab片段的亲和性降低的同时在碱性条件下的化学稳定性提高。这些改善抗体结构域耐碱性的方法都是针对脱酰胺作用和特定氨基酸而开发的。Protein A is a protein molecule with a molecular weight of 42 kDa isolated from the cell wall of Staphylococcus aureus, which contains highly homologous antibody-binding domains E, D, A, B and C. Among the above-mentioned five antibody domains, domain B is particularly advantageous in binding specificity to antibodies. On this basis, the mutated domain Z obtained by mutating the 29-position glycine of domain B to alanine (G29A) not only eliminated the chemical dissociation of domain B caused by deamidation, but also improved its performance in alkaline conditions. Stability under [China Invention Patent Publication No. CN101522278A]. In addition, the binding effect of the domain to the antibody Fab fragment is also reduced with the mutation, and then the binding specificity to the antibody Fc fragment is enhanced [Chinese invention patent, application number: 201080013514.0]. Domain Z and its oligomers have been widely used in protein A chromatography media due to better alkali resistance. The tolerance of domain Z to lye was further improved by N23T mutation [Proteins-Structure Function and Bioinformatics, 2004, 55(2): 407-416]. Patent Publication No. CN1642976A further discloses a method for an immunoglobulin binding protein with at least one asparagine residue mutated, characterized in that the mutations include N23T and N43E, N28A, N6A, N11S, N11S and N23T and N6A and N23T etc. different combinations. But this strategy is only reflected in the modification of a specific amino acid (asparagine). Minakuchi et al. reported that the G29A mutation of antibody domain C can also improve the alkali resistance of domain C [Protein Science, 2013, 22(9): 1230-1238]. Correspondingly, the alkali resistance of G29Y, G29M or G29W mutation in antibody domain C is more advantageous. Kaneka Corporation has disclosed a mutant of antibody domains E, D, A, B and C that mutate glycine into amino acids other than alanine [Publication No. CN 102365361A], which is characterized by the obtained mutant domains The chemical stability under alkaline conditions is improved while the affinity with antibody Fab fragments is reduced. These approaches to improve the alkali resistance of antibody domains were developed for deamidation and specific amino acids.
目前,以结构域Z及其寡聚体为代表的A蛋白色谱配基已经广泛地应用于抗体的纯化过程中。上述色谱过程中包含了利用0.1mol/L氢氧化钠溶液除去色谱介质上残留蛋白质和杂质的CIP步骤。这与前述标准CIP所要求的0.5–1.0mol/L氢氧化钠溶液仍有差距【公开号:CN 101522278A】。标准CIP的策略是将色谱介质暴露于pH高于13的环境中。这对于当前商品化的A蛋白色谱介质而言仍然是非常苛刻的,其会导致A蛋白色谱配基立体结构的破坏乃至丧失以及抗体结合能力的降低【Protein Science,2013,22(9):1230-1238】。因此,进一步提高结构域Z的耐碱性是解决当前A蛋白色谱技术应用安全性和成本的关键。At present, protein A chromatography ligands represented by domain Z and its oligomers have been widely used in the purification process of antibodies. The above chromatographic process includes a CIP step of removing residual proteins and impurities on the chromatographic medium by using 0.1 mol/L sodium hydroxide solution. This is still far from the 0.5–1.0 mol/L sodium hydroxide solution required by the aforementioned standard CIP [Publication No.: CN 101522278A]. The strategy of standard CIP is to expose the chromatographic medium to a pH above 13. This is still very harsh for the current commercial protein A chromatography medium, which will lead to the destruction or even loss of the steric structure of the protein A chromatography ligand and the reduction of antibody binding ability [Protein Science, 2013, 22(9):1230] -1238]. Therefore, further improving the alkali resistance of domain Z is the key to solving the safety and cost of the current protein A chromatography technology application.
除了消除抗体结构域的脱酰胺作用外,解决蛋白质配基结合特异性和化学稳定性的尝试也从其他角度在进行。Deisenhofer通过分析抗体Fc段与A蛋白抗体结构域B的复合物晶体结构,明确了抗体结构域B中11个参与结合的关键氨基酸残基【Biochemistry,1981,20:2361-70】。Palmer等人报道了从链球菌细胞壁分离得到的抗体结合蛋白G分子的一种Y3F/T16I/T18I突变体具有比野生蛋白G更好耐碱性的特征【Journal of Biotechnology,2008,134(3-4):222-230】。李荣秀和李灵舒公开了一种获得低抗体亲和性A蛋白的新方法,其特征在于结构域Z上的F13G/Y14G双突变【公开号:CN 105198973A】。该突变体的抗体亲和性较结构域Z下降90%以上。为了使结构域Z的结合特异性和化学稳定性或者两者之一得到系统地改善,我们在对结构域Z稳定性及其与抗体作用的分子机制深入认知的基础上提出了一个稳定结构域Z的整体策略,通过稳定结构域Z的整体结构获得化学稳定性更好的结构域Z衍生物。In addition to eliminating deamidation of antibody domains, attempts to address binding specificity and chemical stability of protein ligands are also underway from other perspectives. Deisenhofer determined 11 key amino acid residues involved in binding in antibody domain B by analyzing the complex crystal structure of the antibody Fc segment and protein A antibody domain B [Biochemistry, 1981, 20: 2361-70]. Palmer et al. reported that a Y3F/T16I/T18I mutant of an antibody-binding protein G molecule isolated from Streptococcus cell wall had better alkali resistance than wild protein G [Journal of Biotechnology, 2008, 134 (3- 4): 222-230]. Li Rongxiu and Li Lingshu disclosed a new method for obtaining protein A with low antibody affinity, which is characterized by the F13G/Y14G double mutation on domain Z [Publication No.: CN 105198973A]. The antibody affinity of the mutant decreased by more than 90% compared with the domain Z. In order to systematically improve either the binding specificity and chemical stability of domain Z, or both, we propose a stable structure based on an in-depth understanding of domain Z stability and the molecular mechanism of its interaction with antibodies. The overall strategy of domain Z to obtain domain Z derivatives with better chemical stability by stabilizing the overall structure of domain Z.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于改进结构域Z在碱液中的化学稳定性,从而提供一种对抗体具有特异性结合作用结构域Z的衍生物,其在碱性条件下具有良好的化学稳定性;在此基础上,提供利用该结构域Z衍生物为配基的亲和色谱介质制备方法。与以结构域Z及其寡聚体为配基的亲和色谱介质相比,本发明制备的亲和色谱介质的抗体结合能力与之相当。The purpose of the present invention is to improve the chemical stability of domain Z in lye, so as to provide a derivative of domain Z with specific binding to antibodies, which has good chemical stability under alkaline conditions; On this basis, a method for preparing an affinity chromatography medium using the domain Z derivative as a ligand is provided. Compared with the affinity chromatography medium using domain Z and its oligomers as ligands, the antibody binding ability of the affinity chromatography medium prepared by the present invention is comparable.
本发明的目的是通过如下的技术方案得以实现的。The purpose of the present invention is achieved through the following technical solutions.
一种对抗体具有特异性结合作用的A蛋白结构域Z的衍生物,其特征在于所述的结构域Z衍生物由源自SEQ ID No.:1的结构域Z氨基酸序列中除N23T突变以外还有至少1个氨基酸残基发生突变,与亲本分子相比,该结构域Z的衍生物在碱性条件下获得了更高的化学稳定性。A protein A domain Z derivative with specific binding effect on antibodies, characterized in that the domain Z derivative is derived from the domain Z amino acid sequence of SEQ ID No.: 1 except for the N23T mutation At least one amino acid residue was also mutated, and the derivatives of this domain Z obtained higher chemical stability under alkaline conditions compared to the parent molecule.
所述的对抗体具有特异性结合作用的衍生物,其特征在于,结构域Z衍生物可与抗体Fc片段特异性结合。The said derivative with specific binding effect on antibody is characterized in that the domain Z derivative can specifically bind to the Fc fragment of the antibody.
所述的结构域Z衍生物,其特征在于,亲本分子含有SEQ ID No.:1的序列并且氨基酸的突变选自23位天冬酰胺残基以及11位天冬酰胺残基、12位丙氨酸残基、32位谷氨酰胺残基和35位赖氨酸残基中的至少1个氨基酸残基。The said domain Z derivative is characterized in that, the parent molecule contains the sequence of SEQ ID No.: 1 and the amino acid mutation is selected from 23 asparagine residues, 11 asparagine residues, 12 alanine residues At least one amino acid residue among acid residues, glutamine residues at position 32 and lysine residues at position 35.
所述的结构域Z衍生物,其特征在于,其特征在于,在11位天冬酰胺、12位丙氨酸、32位谷氨酰胺和35位赖氨酸已突变,进一步优选为11位天冬酰胺残基突变为苏氨酸、12位丙氨酸残基突变为色氨酸、32位谷氨酰胺残基突变为苏氨酸和35位赖氨酸残基突变为精氨酸。。The said domain Z derivative is characterized in that, it is characterized in that the 11-position asparagine, the 12-position alanine, the 32-position glutamine and the 35-position lysine have been mutated, more preferably the 11-position day The paraparagine residue was mutated to threonine, the alanine residue at position 12 was mutated to tryptophan, the glutamine residue at position 32 was mutated to threonine, and the lysine residue at position 35 was mutated to arginine. .
一种包含2个以上所述的结构域Z衍生物连接而成的多结构域蛋白质,其特征在于,结构域Z衍生物的数量为2–6。A multi-domain protein comprising two or more domain Z derivatives linked together, wherein the number of domain Z derivatives is 2-6.
所述的多结构域蛋白质,其特征在于,各个结构域通过若干个甘氨酸残基连接而成。The multi-domain protein is characterized in that each domain is formed by connecting several glycine residues.
利用本发明的突变结构域Z衍生物为配基的亲和色谱介质制备方法,其特征在于,平均粒径在30–300μm的琼脂糖凝胶粒子孔道内偶联所述任一项的结构域Z衍生物或衍生物多聚体。The method for preparing an affinity chromatography medium using the mutant domain Z derivative of the present invention as a ligand is characterized in that the domain of any one of the above-mentioned domains is coupled in the pores of agarose gel particles with an average particle size of 30-300 μm. Z derivative or derivative multimer.
所述的亲和色谱介质,其特征在于,亲和色谱介质的结构域Z衍生物或衍生物多聚体配基密度为2.0–15mg/mL。The affinity chromatography medium is characterized in that the domain Z derivative or derivative multimer ligand density of the affinity chromatography medium is 2.0-15 mg/mL.
所述的亲和色谱介质,其特征在于,亲和色谱介质可与含有抗体Fc片段的蛋白质结合。The affinity chromatography medium is characterized in that the affinity chromatography medium can bind to the protein containing the Fc fragment of the antibody.
所述的用于抗体分离的亲和色谱介质的方法,其特征在于,结构域Z衍生物或衍生物多聚体通过氨基或羧基偶联于琼脂糖凝胶粒子。The method for the affinity chromatography medium for antibody separation is characterized in that the domain Z derivative or derivative multimer is coupled to the Sepharose particles through amino groups or carboxyl groups.
本发明提供的一种结构域Z的衍生物相较于亲本分子而言在碱性条件下的化学稳定性更好,而且该衍生物的抗体的结合特异性与结构域Z相当。在此基础上制备的以结构域Z衍生物为配基的亲和色谱介质相较于以亲本分子为配基的亲和色谱介质能够耐受更高浓度碱液的CIP清洗并且维持相当的抗体结合能力。Compared with the parent molecule, a derivative of domain Z provided by the present invention has better chemical stability under alkaline conditions, and the binding specificity of the antibody of the derivative is comparable to that of domain Z. The affinity chromatography medium prepared on this basis with domain Z derivatives as ligands can withstand CIP washing with higher concentrations of lye and maintain equivalent antibodies compared to the affinity chromatography medium with parent molecules as ligands binding ability.
本发明提供的一种结构域Z衍生物和以结构域Z衍生物为配基的亲和色谱介质相比现有技术具有如下优点:Compared with the prior art, the domain Z derivative provided by the present invention and the affinity chromatography medium using the domain Z derivative as a ligand have the following advantages:
(1)本发明提供的结构域Z衍生物是基于蛋白结构的整体稳定策略。这一策略的实施是以对结构域Z蛋白结构深刻认知为基础获得的。结构域Z衍生物化学稳定性的提升不局限于序列中单个或者仅仅某种氨基酸残基的突变(如G29A或者天冬酰胺残基的突变等),而是源于结构域Z中Helix 1和2中至少一个氨基酸残基的突变,体现出一种稳定蛋白质分子结构的整体策略。(1) The domain Z derivative provided by the present invention is an overall stabilization strategy based on protein structure. The implementation of this strategy is based on a deep knowledge of the structure of the domain Z protein. The improvement of chemical stability of domain Z derivatives is not limited to the mutation of a single or only a certain amino acid residue in the sequence (such as the mutation of G29A or asparagine residues, etc.), but originates from the
(2)本发明提供的结构域Z衍生物具有更高的化学稳定性。相比于SEQ ID No.:1的结构域Z,序列号为SEQ ID No.:2的衍生物在0.1mol/L氢氧化钠溶液中的蛋白变性温度提高了4℃,序列号为SEQ ID No.:3的衍生物变性温度提高了3.2℃。这一结果显示了本发明提供的结构域Z衍生物具有更高的稳定性。(2) The domain Z derivatives provided by the present invention have higher chemical stability. Compared with the structural domain Z of SEQ ID No.: 1, the protein denaturation temperature of the derivative of SEQ ID No.: 2 in 0.1mol/L sodium hydroxide solution increased by 4°C, and the sequence number is SEQ ID The denaturation temperature of the derivative of No.:3 increased by 3.2℃. This result shows that the domain Z derivatives provided by the present invention have higher stability.
(3)以结构域Z衍生物为配基的亲和色谱介质维持了相当的抗体结合能力。本发明提供的结构域Z衍生物是基于蛋白质分子结构整体稳定策略基础上获得的,氨基酸残基的突变不涉及与抗体Fc片段有结合作用的关键氨基酸残基或者并未改变与抗体Fc片段有结合作用关键氨基酸残基的性质。由此,所获得的结构域Z衍生物基本维持了相当的抗体结合能力和吸附容量。(3) The affinity chromatography medium with domain Z derivatives as ligands maintained comparable antibody binding ability. The domain Z derivatives provided by the present invention are obtained based on the overall stabilization strategy of the protein molecular structure, and the mutation of amino acid residues does not involve the key amino acid residues that bind to the Fc fragment of the antibody or does not change the key amino acid residues that have a binding effect with the Fc fragment of the antibody. Properties of key amino acid residues for binding. Thus, the obtained Domain Z derivatives substantially maintained comparable antibody binding capacity and adsorption capacity.
附图说明Description of drawings
为了使本发明的内容更容易被清楚地理解,下面结合附图,对本发明作进一步详细的说明,其中:In order to make the content of the present invention easier to be understood clearly, the present invention will be described in further detail below in conjunction with the accompanying drawings, wherein:
图1序列号为SEQ ID No.:1-4的A蛋白结构域Z及其衍生物在含100mmol/L氯化钠的20mmol/L磷酸缓冲液和0.1mol/L氢氧化钠溶液中的示差扫描量热结果。Fig. 1 shows the differential expression of protein A domain Z of SEQ ID No. 1-4 and its derivatives in 20 mmol/L phosphate buffer containing 100 mmol/L sodium chloride and 0.1 mol/L sodium hydroxide solution Scan calorimetry results.
图2序列号为SEQ ID No.:5(a)和SEQ ID No.:7(b)的结构域Z衍生物表达产物的离子交换色谱Q Sepharose FF回收情况。Figure 2 shows the recovery of Q Sepharose FF by ion exchange chromatography of the expression products of the domain Z derivatives of SEQ ID No.: 5(a) and SEQ ID No.: 7(b).
图3以结构域Z衍生物为配基的亲和色谱介质在含100mmol/L氯化钠的20mmol/L磷酸缓冲液中抗体吸附结果。Fig. 3 The results of antibody adsorption in 20 mmol/L phosphate buffer containing 100 mmol/L sodium chloride in affinity chromatography medium using domain Z derivatives as ligands.
具体实施方式Detailed ways
本发明实施例仅为解释目的而提供,其不应当解释为限制所附权利要求书定义的本发明。The embodiments of the present invention are provided for illustrative purposes only and should not be construed as limiting the invention as defined by the appended claims.
实施例1序列号为SEQ ID No.:2的结构域Z衍生物的制备和稳定性Example 1 Preparation and stability of domain Z derivatives with SEQ ID No.: 2
利用多肽固相化学合成方法分别合成序列号为SEQ ID No.:1的结构域Z和序列号为SEQ ID No.:2的蛋白质。上述蛋白质溶解于含100mmol/L NaCl的20mmol/L PBS缓冲液(pH 6.0)中,配置成蛋白浓度为1mg/mL的蛋白质溶液。上述两种溶液中蛋白质的变性温度在VP-DSC型示差扫描量热仪中进行。首先,VP-DSC的样品池与参比池经过100mL超纯水清洗之后,使用Hamilton进样器分别吸取经过真空脱气15min以上的缓冲液(含100mmol/L NaCl的20mmol/L PBS缓冲液,pH 6.0)和蛋白质溶液各约0.75mL加入到参比池与样品池中(参比池和样品池的体积为0.5282mL)。多余的溶液经Hamilton进样器移除后,样品池和参比池加盖密封。示差扫描量热测量的温度扫描范围为25—120℃,升温速率为1℃/min。加热开始前,试验温度平衡时间为8min。数据处理经MicroCal-enabled Origin 7.0软件完成。结果如图1所示。结果表明,序列号为SEQ ID No.:1的结构域Z变性温度为75.3℃,序列号为SEQID No.:2的结构域Z衍生物变性温度为79.3℃。这表明,序列号为SEQ ID No.:2的结构域Z衍生物在中性条件下更加稳定。The domain Z with the sequence number of SEQ ID No.: 1 and the protein with the sequence number of SEQ ID No.: 2 were synthesized by solid-phase chemical synthesis of peptides. The above proteins were dissolved in 20 mmol/L PBS buffer (pH 6.0) containing 100 mmol/L NaCl to prepare a protein solution with a protein concentration of 1 mg/mL. The denaturation temperature of proteins in the above two solutions was carried out in a VP-DSC type differential scanning calorimeter. First, after the sample cell and reference cell of VP-DSC were washed with 100 mL of ultrapure water, the buffer solution (20 mmol/L PBS buffer containing 100 mmol/L NaCl, 20 mmol/L PBS buffer containing 100 mmol/L NaCl, pH 6.0) and about 0.75 mL each of the protein solution were added to the reference and sample cells (the volume of the reference and sample cells was 0.5282 mL). After the excess solution was removed by the Hamilton injector, the sample and reference cells were capped and sealed. The temperature scanning range of differential scanning calorimetry was 25-120 °C, and the heating rate was 1 °C/min. Before heating begins, the test temperature equilibration time is 8 min. Data processing was done with MicroCal-enabled Origin 7.0 software. The results are shown in Figure 1. The results showed that the denaturation temperature of the domain Z with the sequence number of SEQ ID No.: 1 was 75.3°C, and the denaturation temperature of the domain Z derivative of the sequence number of SEQ ID No.: 2 was 79.3°C. This indicates that the domain Z derivative of SEQ ID No.: 2 is more stable under neutral conditions.
实施例2序列号为SEQ ID No.:2的结构域Z衍生物在碱液中的稳定性Example 2 The stability of the domain Z derivative of SEQ ID No.: 2 in lye
采用实施例1所述的制备方法合成序列号为SEQ ID No.:1的结构域Z和序列号为SEQ ID No.:2的蛋白质。上述蛋白质溶解于0.1mol/L氢氧化钠溶液中,配置成蛋白浓度为1mg/mL的蛋白质溶液。上述两种溶液中蛋白质的变性温度采用如实施例1中的示差扫描量热方法分析。溶液体系为0.1mol/L氢氧化钠溶液。参比池中注入经过真空脱气15min以上的0.1mol/L氢氧化钠溶液。示差扫描量热测量的温度扫描范围为25—120℃,升温速率为1℃/min。加热开始前,试验温度平衡时间为8min。数据处理经MicroCal-enabled Origin 7.0软件完成。结果如图1所示。结果表明,序列号为SEQ ID No.:1的结构域Z在0.1mol/L氢氧化钠溶液中的变性温度为61.6℃,序列号为SEQ ID No.:2的结构域Z衍生物在0.1mol/L氢氧化钠溶液中的变性温度为64.8℃。这表明,序列号为SEQ ID No.:2的结构域Z衍生物在0.1mol/L氢氧化钠溶液中的化学稳定性更高。The structural domain Z of SEQ ID No.: 1 and the protein of SEQ ID No. 2 were synthesized by the preparation method described in Example 1. The above protein was dissolved in a 0.1 mol/L sodium hydroxide solution to prepare a protein solution with a protein concentration of 1 mg/mL. The denaturation temperature of proteins in the above two solutions was analyzed by differential scanning calorimetry as in Example 1. The solution system is 0.1 mol/L sodium hydroxide solution. The reference cell was injected with 0.1 mol/L sodium hydroxide solution that had been degassed by vacuum for more than 15 min. The temperature scanning range of differential scanning calorimetry was 25-120 °C, and the heating rate was 1 °C/min. Before heating begins, the test temperature equilibration time is 8 min. Data processing was done with MicroCal-enabled Origin 7.0 software. The results are shown in Figure 1. The results showed that the denaturation temperature of domain Z with SEQ ID No. 1 in 0.1 mol/L sodium hydroxide solution was 61.6°C, and the denaturation temperature of domain Z with SEQ ID No.: 2 was 0.1 The denaturation temperature in mol/L sodium hydroxide solution was 64.8°C. This shows that the chemical stability of the domain Z derivative with the sequence number of SEQ ID No.: 2 is higher in 0.1 mol/L sodium hydroxide solution.
实施例3序列号为SEQ ID No.:3的结构域Z衍生物的制备和稳定性Example 3 Preparation and stability of domain Z derivatives with SEQ ID No.: 3
采用实施例1所述的制备方法合成序列号为SEQ ID No.:3的蛋白质。上述蛋白质在中性条件下的变性温度采用如实施例1中的示差扫描量热方法分析。结果如图1所示。结果表明,序列号为SEQ ID No.:3的结构域Z衍生物变性温度为76.9℃。这表明,序列号为SEQID No.:3的结构域Z衍生物在中性条件下比结构域Z更加稳定。The protein with the sequence number of SEQ ID No.: 3 was synthesized by the preparation method described in Example 1. The denaturation temperature of the above proteins under neutral conditions was analyzed by differential scanning calorimetry as in Example 1. The results are shown in Figure 1. The results showed that the denaturation temperature of the domain Z derivative of SEQ ID No.: 3 was 76.9°C. This indicates that the domain Z derivative of SEQ ID No.: 3 is more stable than domain Z under neutral conditions.
实施例4序列号为SEQ ID No.:3的结构域Z衍生物在碱液中的稳定性Example 4 The stability of the domain Z derivative of SEQ ID No.: 3 in lye
序列号为SEQ ID No.:3的结构域Z衍生物在碱液中的稳定性采用实施例2所述方法进行测量。结果如图1所示。结果表明,序列号为SEQ ID No.:3的结构域Z衍生物在0.1mol/L氢氧化钠溶液中的变性温度为62.8℃。这表明,序列号为SEQ ID No.:3的结构域Z衍生物在0.1mol/L氢氧化钠溶液中的化学稳定性更高。The stability of the domain Z derivative of SEQ ID No.: 3 in alkaline solution was measured by the method described in Example 2. The results are shown in Figure 1. The results showed that the denaturation temperature of the domain Z derivative of SEQ ID No.: 3 in 0.1 mol/L sodium hydroxide solution was 62.8°C. This shows that the chemical stability of the domain Z derivative with the sequence number of SEQ ID No.: 3 is higher in 0.1 mol/L sodium hydroxide solution.
实施例5序列号为SEQ ID No.:4的结构域Z衍生物的制备和各种条件下稳定性测量Example 5 Preparation of Domain Z Derivatives with SEQ ID No.: 4 and Measurement of Stability under Various Conditions
采用实施例1所述的制备方法合成序列号为SEQ ID No.:4的蛋白质。上述蛋白质在中性和碱性条件下的变性温度采用如实施例1和2中的示差扫描量热方法分析。结果如图1所示。结果表明,序列号为SEQ ID No.:4的结构域Z衍生物在中性条件下变性温度为77.5℃,在0.1mol/L氢氧化钠溶液中的变性温度为62.4℃。这表明,序列号为SEQ ID No.:4的结构域Z衍生物在中性条件下和0.1mol/L氢氧化钠溶液中的化学稳定性均优于结构域Z。The protein with the sequence number of SEQ ID No.: 4 was synthesized by the preparation method described in Example 1. Denaturation temperatures of the above proteins under neutral and basic conditions were analyzed using differential scanning calorimetry as in Examples 1 and 2. The results are shown in Figure 1. The results showed that the denaturation temperature of the domain Z derivative of SEQ ID No.: 4 was 77.5°C under neutral conditions and 62.4°C in 0.1 mol/L sodium hydroxide solution. This shows that the chemical stability of the domain Z derivative with the sequence number of SEQ ID No.: 4 is better than that of the domain Z under neutral conditions and in 0.1 mol/L sodium hydroxide solution.
实施例6序列号为SEQ ID No.:5的结构域Z衍生物的制备Example 6 Preparation of Domain Z Derivatives with SEQ ID No.: 5
通过化学合成方法设计合成序列号为SEQ ID No.:5的结构域Z衍生物基因序列,其序列SEQ ID No.:6的长度为375bp。上述序列中包括终止密码子TAA以及添加的5’Ndel和3’Xhol。5’Ndel和3’Xhol用于克隆至载体pet-30a(Kan)。用限制性内切酶Ndel和Xhol将载体酶切后连接SEQ ID No.:5基因,获得含有SEQ ID No.:6基因的pet-30a(Kan)质粒。用氯化钙法将质粒转化到BL21DE3细胞中。挑取单菌落的转化BL21DE3大肠杆菌,接种于含有30μg/mL卡那霉素的液体LB培养基中培养过夜后,在无菌条件下接种到发酵液中,接种浓度不低于1:200(v/v),接种后的发酵液在37℃下发酵,直至OD600值达到0.6-0.8时,加入浓度为500mmol/L IPTG至发酵液中IPTG终浓度为0.5mmol/L并继续发酵6h以上。所收集的发酵液经4800rpm离心30min除去上清液后,用20mmol/L Tris-HCl缓冲液(pH7.5)重悬菌体。重复上述操作后,重悬菌体经超声破碎后收集破碎液。细胞破碎液经用0.45μm滤膜过滤后,用Q-Sepharose HP离子交换色谱纯化,吸附缓冲液为20mmol/L Tris-HCl缓冲液(pH7.5),洗脱缓冲液为含1.0mol/L NaCl的20mmol/L Tris-HCl缓冲液(pH7.5)。采用梯度洗脱回收目标蛋白质,梯度条件为30min内洗脱缓冲液线性增至15%。收集在电导率为8-11mS/cm下洗脱的色谱峰。色谱峰结果如图2(a)所示。目标蛋白的纯度为96%。The domain Z derivative gene sequence of SEQ ID No.: 5 was designed and synthesized by chemical synthesis method, and the length of its sequence SEQ ID No.: 6 was 375 bp. The above sequence includes the stop codon TAA and added 5'Ndel and 3'Xhol. 5'Ndel and 3'Xhol were used for cloning into vector pet-30a (Kan). The vector was digested with restriction enzymes Ndel and Xhol, and then ligated with the gene of SEQ ID No.: 5 to obtain the pet-30a (Kan) plasmid containing the gene of SEQ ID No.: 6. The plasmids were transformed into BL21DE3 cells using the calcium chloride method. Pick a single colony of transformed BL21DE3 Escherichia coli, inoculate it in a liquid LB medium containing 30 μg/mL kanamycin and cultivate overnight, then inoculate it into the fermentation broth under sterile conditions, and the inoculation concentration is not less than 1:200 ( v/v), the inoculated fermentation broth was fermented at 37°C until the OD 600 value reached 0.6-0.8, adding IPTG with a concentration of 500 mmol/L to the final concentration of IPTG in the fermentation broth of 0.5 mmol/L and continuing to ferment for more than 6 h . The collected fermentation broth was centrifuged at 4800 rpm for 30 min to remove the supernatant, and the cells were resuspended with 20 mmol/L Tris-HCl buffer (pH 7.5). After repeating the above operations, the resuspended cells were sonicated and the fragmented liquid was collected. The cell fragmentation solution was filtered with a 0.45 μm filter membrane, and purified by Q-Sepharose HP ion exchange chromatography. The adsorption buffer was 20 mmol/L Tris-HCl buffer (pH 7.5), and the elution buffer was 1.0 mol/L. NaCl in 20 mmol/L Tris-HCl buffer (pH 7.5). The target protein was recovered by gradient elution, and the gradient condition was that the elution buffer linearly increased to 15% within 30 min. Chromatographic peaks eluting at conductivities of 8-11 mS/cm were collected. The chromatographic peak results are shown in Figure 2(a). The purity of the target protein was 96%.
实施例7序列号为SEQ ID No.:7的结构域Z衍生物的制备Example 7 Preparation of Domain Z Derivatives with SEQ ID No.: 7
采用实施例6所述的方法,通过化学合成方法设计合成序列号为SEQ ID No.:7的结构域Z衍生物基因序列,其序列SEQ ID No.:8的长度为747bp。用限制性内切酶Ndel和Xhol将载体酶切后连接SEQ ID No.:8基因,获得含有SEQ ID No.:8基因的pet-30a(Kan)质粒。用氯化钙法将质粒转化到BL21DE3细胞中。经微生物发酵和分离纯化,色谱峰结果如图2(b)所示。获得的目标蛋白纯度为95.2%。Using the method described in Example 6, a domain Z derivative gene sequence with the sequence number of SEQ ID No.: 7 was designed and synthesized by chemical synthesis, and the length of the sequence SEQ ID No.: 8 was 747 bp. The vector was digested with restriction enzymes Ndel and Xhol, and then the gene of SEQ ID No.: 8 was ligated to obtain the pet-30a (Kan) plasmid containing the gene of SEQ ID No.: 8. The plasmids were transformed into BL21DE3 cells using the calcium chloride method. After microbial fermentation and separation and purification, the chromatographic peak results are shown in Figure 2(b). The purity of the obtained target protein was 95.2%.
实施例8以SEQ ID No.:2的结构域Z衍生物为配基亲和色谱介质制备Example 8 Preparation of the domain Z derivative of SEQ ID No.: 2 as a ligand affinity chromatography medium
采用实施例1合成的序列号为SEQ ID No.:2的结构域Z衍生物为配基制备亲和色谱介质。制备过程包括介质活化和配基偶联两个步骤。介质活化方法如下:保存于20%乙醇中的Sepharose FF经大量去离子水冲洗和G3漏斗抽干处理后,称量15.0g抽干的介质于100mL锥形瓶中,加入75%(w/w)缩水甘油基三甲基氯化铵(GTMAC)7.5mL,7.5mL双蒸水和0.387g 50%(w/w)氢氧化钠溶液,混合均匀,置于25℃,170rpm下反应过夜;反应结束后,介质经大量去离子水清洗和抽干处理后,称取13.0g抽干介质转移至100mL三角瓶中,加入8.0mL 4.6mol/L NaOH溶液充分悬浮;然后,加入1,4-丁二醇二缩水甘油醚(BDDE)11.0mL,在37℃和170rpm下反应2h;反应结束后用大量去离子水清洗得到环氧基密度为38μmol/g的活化色谱介质。配基偶联方法如下:称取4.0g活化介质置于50mL锥形瓶中,加入10mmol/L碳酸氢钠溶液20mL平衡1h后,抽干溶液,将介质转移至25mL锥形瓶中,加入含40mg序列号为SEQ ID No.:2的结构域Z衍生物的10mmol/L碳酸氢钠溶液8.5mL,充分悬浮后于37℃和170rpm下反应2h;反应结束后介质用大量去离子水清洗并抽干处理;获得的色谱介质置于25mL的锥形瓶中,加入8mL封闭缓冲液,在25℃和170rpm下反应过夜;反应产物依次用含0.15mol/L NaCl的0.1mol/L Tris-HCl缓冲液(pH 8.0)和50mmol/L乙酸缓冲液(pH 4.5)各洗涤三遍,洗涤后的介质最后分别用去离子水和20%的乙醇清洗,获得配基密度为6.3mg/g的亲和色谱介质Z2Sepahrose FF。Affinity chromatography medium was prepared by using the domain Z derivative of SEQ ID No.: 2 synthesized in Example 1 as a ligand. The preparation process includes two steps of media activation and ligand coupling. The medium activation method is as follows: after the Sepharose FF stored in 20% ethanol is rinsed with a large amount of deionized water and dried with a G3 funnel, weigh 15.0 g of the drained medium into a 100 mL conical flask, add 75% (w/w ) glycidyl trimethyl ammonium chloride (GTMAC) 7.5mL, 7.5mL double distilled water and 0.387
采用上述合成方法,加入含40mg序列号为SEQ ID No.:1的结构域Z的10mmol/L碳酸氢钠溶液8.5mL,充分悬浮后于37℃和170rpm下反应2h;获得配基密度为5.7mg/g的亲和色谱介质Z1Sepharose FF。Using the above synthesis method, 8.5 mL of 10 mmol/L sodium bicarbonate solution containing 40 mg of domain Z with the sequence number of SEQ ID No.: 1 was added, fully suspended and reacted at 37° C. and 170 rpm for 2 h; the obtained ligand density was 5.7 mg/g of the affinity chromatography medium Z1Sepharose FF.
在含100mmol/L氯化钠的20mmol/L磷酸盐缓冲液(pH 7.4)中,上述亲和色谱介质Z2Sepharose FF和Z1Sepharose FF的抗体吸附平衡曲线如图3所示,对应的抗体的饱和吸附容量分别为69.0mg/g介质和60.9mg/g介质。In 20mmol/L phosphate buffer (pH 7.4) containing 100mmol/L sodium chloride, the antibody adsorption equilibrium curves of the above affinity chromatography media Z2Sepharose FF and Z1Sepharose FF are shown in Figure 3, and the corresponding saturated adsorption capacities of antibodies 69.0 mg/g medium and 60.9 mg/g medium, respectively.
实施例9以SEQ ID No.:3的结构域Z衍生物为配基亲和色谱介质制备Example 9 Preparation of the domain Z derivative of SEQ ID No.: 3 as ligand affinity chromatography medium
采用实施例8所示的方法制备以SEQ ID No.:3的结构域Z衍生物为配基亲和色谱介质,其中4g活化介质加入含40mg序列号为SEQ ID No.:3的结构域Z衍生物的10mmol/L碳酸氢钠溶液8.0mL,充分悬浮后于37℃和170rpm下反应获得配基密度为7.1mg/g的亲和色谱介质Z3Sepharose FF。在含100mmol/L氯化钠的20mmol/L磷酸盐缓冲液(pH 7.4)中,上述亲和色谱介质的抗体吸附平衡曲线如图3所示,抗体的饱和吸附容量为52.3mg/g介质。The method shown in Example 8 was used to prepare the domain Z derivative of SEQ ID No.: 3 as the ligand affinity chromatography medium, wherein 4 g of the activation medium was added to contain 40 mg of the domain Z of SEQ ID No.: 3. 8.0 mL of a 10 mmol/L sodium bicarbonate solution of the derivative was fully suspended and reacted at 37° C. and 170 rpm to obtain an affinity chromatography medium Z3Sepharose FF with a ligand density of 7.1 mg/g. In 20mmol/L phosphate buffer (pH 7.4) containing 100mmol/L sodium chloride, the antibody adsorption equilibrium curve of the above affinity chromatography medium is shown in Figure 3, and the saturated adsorption capacity of the antibody is 52.3mg/g medium.
实施例10以SEQ ID No.:4的结构域Z衍生物为配基亲和色谱介质制备Example 10 Preparation of the domain Z derivative of SEQ ID No.: 4 as ligand affinity chromatography medium
采用实施例8所述方法制备以SEQ ID No.:4的结构域Z衍生物为配基亲和色谱介质,其中4g活化介质加入含100mg序列号为SEQ ID No.:4的结构域Z衍生物的10mmol/L碳酸氢钠溶液8.0mL,充分悬浮后于37℃和170rpm下反应获得配基密度为14.8mg/g的亲和色谱介质Z4Sepharose FF。在含100mmol/L氯化钠的20mmol/L磷酸盐缓冲液(pH 7.4)中,上述亲和色谱介质的抗体吸附平衡曲线如图3所示,抗体的饱和吸附容量为45.8mg/g介质。The method described in Example 8 was used to prepare the domain Z derivative of SEQ ID No.: 4 as the ligand affinity chromatography medium, wherein 4 g of the activation medium was added with 100 mg of the domain Z derivative of SEQ ID No.: 4. 8.0 mL of a 10 mmol/L sodium bicarbonate solution of the compound was fully suspended and reacted at 37 °C and 170 rpm to obtain an affinity chromatography medium Z4Sepharose FF with a ligand density of 14.8 mg/g. In 20mmol/L phosphate buffer (pH 7.4) containing 100mmol/L sodium chloride, the antibody adsorption equilibrium curve of the above affinity chromatography medium is shown in Figure 3, and the saturated adsorption capacity of the antibody is 45.8mg/g medium.
实施例11以SEQ ID No.:5的结构域Z衍生物为配基亲和色谱介质制备Example 11 Preparation of Affinity Chromatography Medium Using Domain Z Derivative of SEQ ID No.: 5 as Ligand
采用实施例8所述方法制备以SEQ ID No.:5的结构域Z衍生物为配基亲和色谱介质,其中介质活化过程中加入75%(w/w)缩水甘油基三甲基氯化铵(GTMAC)20.0mL,1,4-丁二醇二缩水甘油醚(BDDE)的加入量为20.0mL,反应得到环氧基密度为77.1μmol/g的活化色谱介质。配基偶联过程中,4.0g活化介质中加入含20mg根据实施例5所制备的序列号为SEQID No.:5的结构域Z衍生物的10mmol/L碳酸氢钠溶液8.5mL,反应获得配基密度为2.3mg/g的亲和色谱介质Z5Sepahrose FF。在含100mmol/L氯化钠的20mmol/L磷酸盐缓冲液(pH7.4)中,上述亲和色谱介质的抗体吸附平衡曲线如图3所示,抗体的饱和吸附容量为31.1mg/g介质。The method described in Example 8 was used to prepare the domain Z derivative of SEQ ID No.: 5 as the ligand affinity chromatography medium, wherein 75% (w/w) glycidyl trimethyl chloride was added during the medium activation process. Ammonium (GTMAC) 20.0 mL, 1,4-butanediol diglycidyl ether (BDDE) was added in an amount of 20.0 mL, and the reaction obtained an activated chromatography medium with an epoxy group density of 77.1 μmol/g. During the ligand coupling process, 8.5 mL of 10 mmol/L sodium bicarbonate solution containing 20 mg of the domain Z derivative with the sequence number of SEQ ID No.: 5 prepared according to Example 5 was added to 4.0 g of the activation medium, and the reaction obtained the ligand. The affinity chromatography medium Z5 Sepahrose FF with a base density of 2.3 mg/g. In 20mmol/L phosphate buffer (pH7.4) containing 100mmol/L sodium chloride, the antibody adsorption equilibrium curve of the above affinity chromatography medium is shown in Figure 3, and the saturated adsorption capacity of the antibody is 31.1mg/g medium .
实施例12以SEQ ID No.:7的结构域Z衍生物为配基亲和色谱介质制备Example 12 Preparation of the domain Z derivative of SEQ ID No.: 7 as ligand affinity chromatography medium
采用实施例9所述方法制备以SEQ ID No.:7的结构域Z衍生物为配基亲和色谱介质,其中配基偶联过程中,4.0g活化介质中加入含30mg根据实施例7所制备的序列号为SEQID No.:7的结构域Z衍生物的10mmol/L碳酸氢钠溶液8.5mL,反应获得配基密度为6.6mg/g的亲和色谱介质Z7Sepahrose FF。在含100mmol/L氯化钠的20mmol/L磷酸盐缓冲液(pH7.4)中,上述亲和色谱介质的抗体吸附平衡曲线如图3所示,抗体的饱和吸附容量为72.7mg/g介质。The method described in Example 9 was used to prepare the domain Z derivative of SEQ ID No.: 7 as the ligand affinity chromatography medium. During the ligand coupling process, 4.0 g of the activation medium was added with 30 mg of 8.5 mL of a 10 mmol/L sodium bicarbonate solution of the prepared domain Z derivative of SEQ ID No.: 7 was prepared and reacted to obtain an affinity chromatography medium Z7 Sepahrose FF with a ligand density of 6.6 mg/g. In 20mmol/L phosphate buffer (pH7.4) containing 100mmol/L sodium chloride, the antibody adsorption equilibrium curve of the above affinity chromatography medium is shown in Figure 3, and the saturated adsorption capacity of the antibody is 72.7mg/g medium .
本发明公开和提出的一种稳定性改良的葡萄球菌A蛋白结构域Z衍生物及其应用,本领域技术人员可通过借鉴本文内容,适当改变条件路线等环节实现,尽管本发明的方法和制备技术已通过较佳实施例子进行了描述,相关技术人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和技术路线进行改动或重新组合,来实现最终的制备技术。特别需要指出的是,所有相类似的替换和改动对本领域技术人员来说是显而易见的,他们都被视为包括在本发明精神、范围和内容中。A staphylococcal protein A domain Z derivative with improved stability disclosed and proposed in the present invention and its application can be realized by those skilled in the art by referring to the content of this article and appropriately changing the conditional route and other links, although the method and preparation of the present invention The technology has been described by preferred embodiments, and those skilled in the art can obviously modify or recombine the methods and technical routes described herein without departing from the content, spirit and scope of the present invention to achieve the final preparation technology. It should be particularly pointed out that all similar substitutions and modifications apparent to those skilled in the art are deemed to be included in the spirit, scope and content of the present invention.
SEQ ID No.:1SEQ ID No.: 1
SEQ ID No.:2SEQ ID No.: 2
SEQ ID No.:3SEQ ID No.: 3
SEQ ID No.:4SEQ ID No.: 4
SEQ ID No.:5SEQ ID No.: 5
SEQ ID No.:6SEQ ID No.: 6
CATATGGTGGATAACAAGTTCAATAAAGAGCAGCAGACCATTTTTTATGAAATTCTGCATCTGCCGAATCTGACCGAAGAACAGCGCAACGCCTTCATCACCAGTCTGCGTGATGATCCGAGCCAGAGCGCCAATCTGCTGGCCGAAGCCAAAAAGCTGAACGATGCCCAGGCACCGAAGG GCGGTGGCGGTG TGGACAATAAGTTTAACAAAGAGCAACAAACCATCTTCTACGAAATCCTGCACCTGCCGAACCTGACCGAGGAACAACGCAATGCCTTCATTACCAGCCTGCGTGATGACCCGAGCCAAAGTGCCAACCTGCTGGCCGAAGCCAAGAAGCTGAATGATGCCCAAGCCCCGAAATAACTC GAG CATATG GTGGATAACAAGTTCAATAAAGAGCAGCAGACCATTTTTTATGAAATTCTGCATCTGCCGAATCTGACCGAAGAACAGCGCAACGCCTTCATCACCAGTCTGCGTGATGATCCGAGCCAGAGCGCCAATCTGCTGGCCGAAGCCAAAAAGCTGAACGATGCCCAGGCACCGAAGG GCGGTGGCGGTG TGGACAATAAGTTTAACAAAGAGCAACAAACCATCTTCTACGAAATCCTGCACCTGCCGAACCTGACCGAGGAACAACGCAATGCCTTCATTACCAGCCTGCGTGATGACCCGAGCCAAAGTGCCAACCTGCTGGCCGAAGCCAAGAAGCTGAATGATGCCCAAGCCCCGAAATAA CTC GAG
SEQ ID No.:7SEQ ID No.: 7
SEQ ID No.:8SEQ ID No.: 8
CATATGGTGGATAACAAGTTCAATAAAGAGCAGCAGACCATTTTTTATGAAATTCTGCATCTGCCGAATCTGACCGAAGAACAGCGCAACGCCTTCATCACCAGTCTGCGTGATGATCCGAGCCAGAGCGCCAATCTGCTGGCCGAAGCCAAAAAGCTGAACGATGCCCAGGCACCGAAGGGCGGTGGCGGTGTGGACAATAAGTTTAACAAAGAGCAACAAACCATCTTCTACGAAATCCTGCACCTGCCGAACCTGACCGAGGAACAACGCAATGCCTTCATTACCAGCCTGCGTGATGACCCGAGCCAAAGTGCCAACCTGCTGGCCGAAGCCAAGAAGCTGAATGATGCCCAAGCCCCGAAAGGTGGCGGTGGTGTTGATAATAAATTTAATAAGGAACAACAGACCATCTTTTATGAAATCCTGCATCTGCCGAACCTGACAGAGGAGCAGCGCAACGCCTTTATCACCAGTCTGCGCGACGATCCGAGCCAAAGCGCCAATCTGCTGGCCGAGGCCAAGAAACTGAATGATGCACAGGCACCTAAAGGCGGTGGCGGCGTGGACAACAAATTTAACAAGGAACAGCAGACCATCTTCTATGAGATCCTGCACCTGCCGAATCTGACCGAAGAACAGCGCAATGCCTTTATTACCAGCCTGCGCGATGATCCTAGCCAGAGTGCCAACCTGCTGGCCGAAGCAAAAAAGCTGAATGACGCCCAGGCCCCGAAGTAACTCGAG CATATG CTCGAG
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