CN111249771A - Preparation method and regeneration method of chromatographic column for purifying gram-grade mechanical functional protein on large scale - Google Patents
Preparation method and regeneration method of chromatographic column for purifying gram-grade mechanical functional protein on large scale Download PDFInfo
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- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims abstract description 97
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 86
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- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 12
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- 229910000363 nickel(II) sulfate Inorganic materials 0.000 claims description 3
- 238000007781 pre-processing Methods 0.000 claims description 3
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- CYWWMFJMFFDFHD-UHFFFAOYSA-N Cl.[O-2].[Na+].[Na+] Chemical compound Cl.[O-2].[Na+].[Na+] CYWWMFJMFFDFHD-UHFFFAOYSA-N 0.000 claims 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 claims 1
- 239000001257 hydrogen Substances 0.000 claims 1
- 239000000945 filler Substances 0.000 abstract description 20
- 238000010828 elution Methods 0.000 abstract description 12
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 10
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- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
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- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 1
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- 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
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
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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
- C07K1/18—Ion-exchange chromatography
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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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- Life Sciences & Earth Sciences (AREA)
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- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
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Abstract
本申请属于蛋白纯化技术领域，公开了一种大批量纯化克级力学功能蛋白的层析柱的制备方法及再生方法。本发明所述大批量纯化克级力学功能蛋白的层析柱的制备方法采用中、高流速将大体积镍亲和层析柱料或阳离子交换层析柱料填充至大容量柱管中。本发明所述制备方法得到的层析柱结合蛋白量大，一次可纯化出克级蛋白，纯度及回收率均达到了预装柱水准，同时可以有效减少柱效的损耗且有效减少洗脱峰拖尾现象。本发明所述纯化力学功能蛋白的层析柱的再生方法可快速去除纯化力学功能蛋白后层析柱上的残留杂质，延长填料寿命，降低使用成本并保证蛋白质量稳定，提高生产效率。且再生效果极佳，DNA、蛋白、内毒素、有机碳的残留非常低。The application belongs to the technical field of protein purification, and discloses a preparation method and a regeneration method of a chromatography column for purifying gram-level mechanically functional proteins in large quantities. The method for preparing a chromatography column for bulk purification of gram-level mechanically functional proteins of the present invention uses medium and high flow rates to fill a large volume of nickel affinity chromatography column material or cation exchange chromatography column material into a large volume column tube. The chromatographic column obtained by the preparation method of the present invention has a large amount of bound protein, gram-level protein can be purified at one time, and the purity and recovery rate have reached the level of prepacked columns, and at the same time, the loss of column efficiency can be effectively reduced and the elution peak can be effectively reduced trailing phenomenon. The method for regenerating a chromatography column for purifying mechanically functional protein of the present invention can rapidly remove residual impurities on the chromatography column after purifying mechanically functional protein, prolong the life of the filler, reduce the use cost, ensure stable protein quality, and improve production efficiency. And the regeneration effect is excellent, and the residue of DNA, protein, endotoxin and organic carbon is very low.
Description
技术领域technical field
本发明属于蛋白纯化技术领域，具体涉及一种大批量纯化克级力学功能蛋白的层析柱的制备方法及再生方法。The invention belongs to the technical field of protein purification, and in particular relates to a preparation method and a regeneration method of a chromatography column for purifying gram-level mechanical functional protein in large quantities.
背景技术Background technique
力学功能蛋白，为一类具有一定力学强度的功能蛋白，如蛛丝弹性蛋白、节肢弹性蛋白、丝心蛋白、贻贝类蛋白等。力学功能蛋白在生物医学材料如蛋白纤维，生物胶水，药物载体等方面具有广泛应用。申请人构建此类蛋白过程中加入了6×His标签，可用亲和层析及离子交换层析柱对蛋白进行纯化。但力学功能蛋白结构复杂，大部分含有大量的β片层结构，且具有100KD以上的分子量，纯化较为困难，而且由于其氨基酸组成较为复杂，且较为容易与金属离子配位，导致其在层析柱上较难彻底去除。因此，力学功能蛋白的纯化不仅仅需要特殊装填的蛋白层析柱，同时还需要特殊的层析柱再生方式来延长层析柱的寿命。Mechanical functional proteins are a class of functional proteins with certain mechanical strength, such as spider silk elastin, arthropod elastin, silk fibroin, and mussel-like proteins. Mechanically functional proteins have a wide range of applications in biomedical materials such as protein fibers, biological glues, and drug carriers. The applicant added a 6×His tag in the process of constructing such a protein, and the protein can be purified by affinity chromatography and ion exchange chromatography. However, the mechanical functional proteins are complex in structure, most of them contain a large number of β-sheet structures, and have a molecular weight of more than 100KD, making it difficult to purify, and because of their complex amino acid composition and easy coordination with metal ions, they are difficult to chromatograph. It is difficult to remove completely from the column. Therefore, the purification of mechanically functional proteins not only requires a special packed protein chromatography column, but also requires a special chromatography column regeneration method to prolong the life of the chromatography column.
目前，层析柱主要分为预装柱及组装柱，预装柱是指一定体积的填充好相关层析柱料的层析柱，其优点在于蛋白吸附效果好且工艺较为稳定，但是，其价格较为高昂且体积较为固定，一般为5ml-50ml，不适用于大规模纯化。更重要的是，力学功能蛋白对层析柱柱料的损伤较大，最终导致成本增高，因此，预装柱不适合力学功能蛋白纯化。At present, chromatography columns are mainly divided into prepacked columns and assembled columns. Prepacked columns refer to a certain volume of chromatography columns filled with relevant chromatography column materials. The advantages are that the protein adsorption effect is good and the process is relatively stable. However, its The price is relatively high and the volume is relatively fixed, generally 5ml-50ml, which is not suitable for large-scale purification. What's more, the mechanically functional protein has great damage to the column material, which eventually leads to higher cost. Therefore, the prepacked column is not suitable for the purification of mechanically functional protein.
组装层析柱是指将指定亲和层析柱料填入到空的玻璃柱管中，其优点在于可以根据实验规模及所纯化蛋白特性灵活确定层析柱体积及层析介质，但是，由于其在纯化仪上在线填充，因此不同的填充流速及不同的层析柱管都会对后续的纯化效果造成一定的影响。因此建立了一种适合于力学功能蛋白纯化的层析柱装填方法，确定适合于力学功能蛋白的层析柱体积及装填流速，对大批量纯化克级力学功能蛋白具有重要意义。Assembling a chromatography column refers to filling the specified affinity chromatography column material into an empty glass column tube. The advantage is that the column volume and chromatography medium can be flexibly determined according to the experimental scale and the characteristics of the purified protein. It is filled online on the purifier, so different filling flow rates and different chromatography column tubes will have a certain impact on the subsequent purification effect. Therefore, a chromatography column packing method suitable for the purification of mechanically functional proteins was established, and the determination of the column volume and packing flow rate suitable for mechanically functional proteins was of great significance for the large-scale purification of gram-level mechanically functional proteins.
然而，组装层析柱在纯化力学功能蛋白后，填料会残留一些黄色及粘性物质，经鉴定为残留的DNA和截断表达蛋白等物质，会对下次使用造成极大影响，大幅度缩短纯化柱的使用寿命，这对用大体积层析柱纯化的力学功能蛋白来说，会大幅度增加成本。更严重的是，残留物质会导致纯化产品质量大幅度下降。而现有的层析柱再生方式主要用碱进行浸泡冲洗，这种方法并不能彻底清洗纯化力学功能蛋白后残留的杂质，尤其是残留的高电荷密度蛋白及DNA。However, after purifying the mechanically functional protein of the assembled chromatography column, some yellow and sticky substances will remain in the filler, which are identified as residual DNA and truncated expressed proteins, which will have a great impact on the next use and greatly shorten the purification column. Longevity of life, which will greatly increase the cost for mechanically functional proteins purified by large-volume chromatography columns. More seriously, residual substances can lead to a substantial decline in the quality of the purified product. However, the existing chromatographic column regeneration method mainly uses alkali to soak and rinse, and this method cannot thoroughly clean the residual impurities after purification of mechanically functional proteins, especially the residual high charge density protein and DNA.
发明内容SUMMARY OF THE INVENTION
有鉴于此，本发明的目的在于针对现有技术中存在的缺陷，提供一种大批量纯化克级力学功能蛋白的层析柱的制备方法及再生方法。In view of this, the purpose of the present invention is to provide a method for preparing and regenerating a chromatography column for purifying gram-level mechanically functional protein in large quantities in view of the defects existing in the prior art.
为实现本发明的目的，本发明采用如下技术方案：For realizing the purpose of the present invention, the present invention adopts following technical scheme:
一种大批量纯化克级力学功能蛋白的层析柱的制备方法，采用中、高流速将大体积镍亲和层析柱料或阳离子交换层析柱料填充至大容量柱管中。The invention discloses a method for preparing a chromatography column for purifying gram-level mechanical functional proteins in large quantities. A large-volume nickel affinity chromatography column material or a cation-exchange chromatography column material is filled into a large-capacity column tube by adopting medium and high flow rates.
本发明经过不同的条件筛选，本发明首先确定了适合于力学功能蛋白的层析柱体积及装填流速，建立一种适合于力学功能蛋白纯化的层析柱装填方法。采用本发明所述制备方法可以得到能够纯化出90％纯度以上的克级力学功能蛋白的亲和层析柱，且对柱效损失较少。采用得到的亲和层析柱可以大批量对力学功能蛋白进行纯化，得到纯度较高的蛋白。After screening under different conditions, the present invention first determines the chromatography column volume and packing flow rate suitable for the mechanically functional protein, and establishes a chromatography column packing method suitable for the purification of the mechanically functional protein. By using the preparation method of the present invention, an affinity chromatography column capable of purifying a gram-level mechanical functional protein with a purity of more than 90% can be obtained, and the loss of column efficiency is less. Using the obtained affinity chromatography column, the mechanically functional protein can be purified in large quantities, and the protein with higher purity can be obtained.
在本发明中，所述的大容量柱管是指内径为50mm的双层钢化玻璃套管，最大承受压力为0.5MPa，容积为559ml。In the present invention, the large-capacity column tube refers to a double-layer tempered glass sleeve with an inner diameter of 50 mm, a maximum pressure of 0.5 MPa, and a volume of 559 ml.
在本发明中，所述的镍亲和层析柱填料型号为Ni Sepharose 6 FF，所述阳离子交换层析柱填料型号为SP Sepharose HP。In the present invention, the type of the filler for the nickel affinity chromatography column is Ni Sepharose 6 FF, and the type of the filler for the cation exchange chromatography column is SP Sepharose HP.
在具体实施方案中，所述的大体积Sepharose Ni 6xFF柱料或SP HP柱料其特定体积为500ml。In a specific embodiment, the specific volume of the large volume Sepharose Ni 6xFF column material or SP HP column material is 500ml.
在本发明中，对于镍亲和层析柱，所述的中、高流速为填充流速40ml/min，固定流速80ml/min。In the present invention, for the nickel affinity chromatography column, the medium and high flow rates are the filling flow rate of 40ml/min and the fixed flow rate of 80ml/min.
在本发明中，对于阳离子交换层析柱，所述的中、高流速为填充流速为25ml/min，固定流速为50ml/min。In the present invention, for the cation exchange chromatography column, the medium and high flow rates are that the filling flow rate is 25ml/min, and the fixed flow rate is 50ml/min.
进一步的，在本发明中，所述的镍亲和层析柱料或阳离子交换层析柱料装填方法主要包括以下几个步骤：Further, in the present invention, the described nickel affinity chromatography column material or cation exchange chromatography column material packing method mainly includes the following steps:
(1)柱料准备：将柱料用超纯水清洗两到三遍，确保其中的乙醇被彻底除去；(1) Preparation of column material: Clean the column material two to three times with ultrapure water to ensure that the ethanol therein is completely removed;
(2)柱管准备：检查所用柱管密封性，润湿柱管底部，静置待用；(2) Preparation of the column tube: check the tightness of the column tube used, wet the bottom of the column tube, and let it stand for use;
(3)平衡：将处理好的柱料沿一侧倒入柱管中，保证柱管垂直于水平面；同时将柱子顶端连接头连接至纯化系统中；插入适配柱并密封；打开系统并调整流速至中流速直到柱料不再下降；(3) Balance: Pour the treated column material into the column tube along one side to ensure that the column tube is perpendicular to the horizontal plane; at the same time, connect the top connector of the column to the purification system; insert the adapter column and seal it; open the system and adjust Flow rate to medium flow rate until the column material no longer falls;
(4)装柱：等柱料不再下降后，调整流速至高流速，运行30-45min，标记此时柱料位置，下移适配柱至标记位置后再下移1.5-3mm，密封；(4) Column loading: After the column material no longer descends, adjust the flow rate to a high flow rate, run for 30-45min, mark the position of the column material at this time, move the adapter column down to the marked position and then move it down 1.5-3mm, and seal;
(5)平衡：用5cv的平衡缓冲液冲洗层析柱，确保系统中A280，254，电导值平衡；(5) Equilibration: rinse the column with 5 cv equilibration buffer to ensure that the A280, 254, and conductance values in the system are balanced;
(6)保存：用20％乙醇水溶液冲洗层析柱，之后竖直保存。(6) Storage: The chromatography column was washed with 20% ethanol aqueous solution, and then stored vertically.
在本发明中，对于镍亲和层析柱，步骤(4)中所述平衡缓冲液为pH8.0的含有0.5M NaCl、0.02M咪唑的0.05M磷酸钠缓冲液。In the present invention, for the nickel affinity chromatography column, the equilibration buffer in step (4) is a 0.05M sodium phosphate buffer containing 0.5M NaCl and 0.02M imidazole at pH 8.0.
在本发明中，对于阳离子交换层析柱，步骤(4)中所述平衡缓冲液为pH8.0的含有0.05M NaCl的0.05M磷酸钠缓冲液。In the present invention, for the cation exchange chromatography column, the equilibration buffer in step (4) is 0.05M sodium phosphate buffer containing 0.05M NaCl at pH 8.0.
本发明还提供了所述大批量纯化克级力学功能蛋白的层析柱的再生方法。The present invention also provides a method for regenerating the chromatography column for purifying the gram-level mechanically functional protein in large quantities.
本发明所述大批量纯化克级力学功能蛋白的层析柱的再生方法具体包括如下步骤：The method for regenerating a chromatography column for purifying gram-level mechanically functional proteins in large quantities according to the present invention specifically includes the following steps:
(A)将层析柱进行预处理；(A) preprocessing the chromatography column;
(B)用氢氧化钠溶液或含有高浓度氯化钠的氢氧化钠溶液清洗所述预处理后的层析柱，然后用去离子水清洗层析柱，去掉所述层析柱残留清洗液；(B) use sodium hydroxide solution or the sodium hydroxide solution that contains high concentration sodium chloride to wash the chromatography column after described pretreatment, then wash the chromatography column with deionized water, remove the residual cleaning solution of the chromatography column ;
(C)用pH为4.0-5.0的含有TritonX-100的乙二胺四乙酸水溶液清洗所述层析柱；(C) washing the chromatography column with an aqueous solution of EDTA containing TritonX-100 at a pH of 4.0-5.0;
(D)用去离子水清洗层析柱，去掉所述层析柱残留清洗液。(D) Washing the chromatography column with deionized water to remove the residual cleaning solution of the chromatography column.
在本发明中，所述步骤(A)中所述预处理具体为所述镍亲和层析柱用脱镍缓冲液冲洗后用水冲洗；所述阳离子交换层析柱用水冲洗。In the present invention, the pretreatment in the step (A) is specifically that the nickel affinity chromatography column is washed with a de-nickel buffer solution and then washed with water; the cation exchange chromatography column is washed with water.
进一步的，在本发明中，所述镍亲和层析柱用脱镍缓冲液为含有50mM EDTA、pH8.0的20mM磷酸钠缓冲液。Further, in the present invention, the nickel-removing buffer for the nickel affinity chromatography column is a 20 mM sodium phosphate buffer containing 50 mM EDTA and pH 8.0.
在本发明中，所述步骤(A)中所述预处理时所述脱镍缓冲液冲洗的流速为40ml/min。In the present invention, the flow rate of the de-nickel buffer flushing during the pretreatment in the step (A) is 40 ml/min.
在本发明中，所述步骤(B)中针对所述镍亲和层析柱用氢氧化钠溶液清洗所述预处理后的层析柱，所述氢氧化钠溶液其浓度为1M。In the present invention, in the step (B), the pretreated column is washed with sodium hydroxide solution for the nickel affinity chromatography column, and the concentration of the sodium hydroxide solution is 1M.
在本发明中，所述步骤(B)中针对所述阳离子交换层析柱用含有高浓度氯化钠的氢氧化钠溶液其为含0.5-2M氯化钠的0.2-1M氢氧化钠溶液。在一些实施方案中，所述含有高浓度氯化钠的氢氧化钠溶液其为含2M氯化钠的0.5M氢氧化钠溶液。In the present invention, the sodium hydroxide solution containing high concentration sodium chloride is used for the cation exchange chromatography column in the step (B), which is a 0.2-1M sodium hydroxide solution containing 0.5-2M sodium chloride. In some embodiments, the sodium hydroxide solution containing high concentration of sodium chloride is a 0.5M sodium hydroxide solution containing 2M sodium chloride.
在本发明中，所述步骤(C)中所述含有TritonX-100的乙二胺四乙酸水溶液其浓度为0.5-2％体积浓度的TritongX-100和0.1-0.3M的乙二胺四乙酸。In the present invention, the concentration of the EDTA aqueous solution containing TritonX-100 in the step (C) is 0.5-2% volume concentration of TritongX-100 and 0.1-0.3M EDTA.
在一些实施方案中，所述含有TritonX-100的乙二胺四乙酸水溶液其浓度为1％体积浓度的TritongX-100和0.2M的乙二胺四乙酸水溶液，其pH为4.5。In some embodiments, the aqueous solution of EDTA containing TritonX-100 has a concentration of 1% by volume of TritongX-100 and a 0.2M aqueous solution of EDTA, and its pH is 4.5.
进一步的，所述步骤(D)后镍亲和层析柱还包括用0.1M的硫酸镍缓冲液冲洗上镍的步骤。Further, after the step (D), the nickel affinity chromatography column further includes a step of rinsing nickel with 0.1M nickel sulfate buffer.
由上述技术方案可知，本发明提供了一种大批量纯化克级力学功能蛋白的层析柱的制备方法及再生方法。本发明所述大批量纯化克级力学功能蛋白的层析柱的制备方法采用中、高流速将大体积镍亲和层析柱料或阳离子交换层析柱料填充至大容量柱管中。本发明所述制备方法得到的层析柱结合蛋白量大，一次可纯化出克级蛋白，纯度及回收率均达到了预装柱水准，同时可以有效减少柱效的损耗且有效减少洗脱峰拖尾现象。本发明所述纯化力学功能蛋白的层析柱的再生方法可快速去除纯化力学功能蛋白后层析柱上的残留杂质，延长填料寿命，降低使用成本并保证蛋白质量稳定，提高生产效率。且再生效果极佳，DNA、蛋白、内毒素、有机碳的残留非常低。As can be seen from the above technical solutions, the present invention provides a preparation method and a regeneration method of a chromatography column for purifying gram-level mechanical functional proteins in large quantities. The method for preparing a chromatography column for bulk purification of gram-level mechanically functional proteins of the present invention uses medium and high flow rates to fill a large volume of nickel affinity chromatography column material or cation exchange chromatography column material into a large volume column tube. The chromatographic column obtained by the preparation method of the present invention has a large amount of bound protein, gram-level protein can be purified at one time, and the purity and recovery rate have reached the level of prepacked columns, and at the same time, the loss of column efficiency can be effectively reduced and the elution peak can be effectively reduced trailing phenomenon. The method for regenerating a chromatography column for purifying mechanically functional protein of the present invention can rapidly remove residual impurities on the chromatography column after purifying mechanically functional protein, prolong the life of the filler, reduce the use cost, ensure stable protein quality, and improve production efficiency. And the regeneration effect is excellent, and the residue of DNA, protein, endotoxin and organic carbon is very low.
附图说明Description of drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art.
图1示大容量层析柱示意图；Figure 1 shows a schematic diagram of a large-capacity chromatography column;
图2示力学功能蛋白纯化电泳对比图(镍柱)，蛋白纯度>90％；Figure 2 shows the electrophoresis comparison chart of mechanical functional protein purification (nickel column), and the protein purity is >90%;
图3示力学功能蛋白纯化电泳对比图(离子交换)，蛋白纯度>96％；Figure 3 shows the electrophoresis comparison chart of mechanical functional protein purification (ion exchange), and the protein purity is >96%;
图4示力学功能蛋白纯化效果UV280峰图(本发明镍亲和层析柱)；Fig. 4 shows the UV280 peak diagram of mechanical functional protein purification effect (nickel affinity chromatography column of the present invention);
图5示力学功能蛋白纯化效果UV280峰图(本发明SP层析柱)；Fig. 5 shows the UV280 peak diagram of the purification effect of mechanical functional protein (SP chromatography column of the present invention);
图6示力学功能蛋白纯化效果UV280峰图(商业镍亲和层析预装柱)；Figure 6 shows the UV280 peak diagram of the purification effect of mechanically functional protein (commercial nickel affinity chromatography prepacked column);
图7示力学功能蛋白纯化效果UV280峰图(商业离子交换层析预装柱)。Figure 7 shows the UV280 peak diagram of the purification effect of mechanically functional protein (commercial ion exchange chromatography prepacked column).
具体实施方式Detailed ways
本发明公开了一种大批量纯化克级力学功能蛋白的层析柱的制备方法及再生方法。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及产品已经通过较佳实施例进行了描述，相关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进行改动或适当变更与组合，来实现和应用本发明技术。The invention discloses a preparation method and a regeneration method of a chromatography column for purifying gram-level mechanical functional protein in large quantities. Those skilled in the art can learn from the content of this document and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and product of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention. Invention technology.
为实现本发明的目的，本发明采用如下技术方案：For realizing the purpose of the present invention, the present invention adopts following technical scheme:
一种大批量纯化克级力学功能蛋白的层析柱的制备方法，采用中、高流速将大体积镍亲和层析柱料或阳离子交换层析柱料填充至大容量柱管中。The invention discloses a method for preparing a chromatography column for purifying gram-level mechanical functional proteins in large quantities. A large-volume nickel affinity chromatography column material or a cation-exchange chromatography column material is filled into a large-capacity column tube by adopting medium and high flow rates.
本发明通过对装柱子过程中条件的探索，用一定中高流速对装有大体积Sepharose Ni 6xFF柱料或SP HP的大容量柱管进行填充，最终得到能够纯化出90％纯度以上的克级力学功能蛋白的亲和层析柱，且对柱效损失较少。By exploring the conditions in the column packing process, the invention fills the large-capacity column tube with the large-volume Sepharose Ni 6xFF column material or SP HP with a certain medium and high flow rate, and finally obtains gram-level mechanics that can purify more than 90% purity. Affinity chromatography column for functional proteins with less loss of column efficiency.
进一步的，在本发明中，所述的镍亲和层析柱料装填方法主要包括以下几个步骤：Further, in the present invention, the described nickel affinity chromatography column material packing method mainly includes the following steps:
(1)柱料准备：将500ml Sepharose Ni 6xFF柱料用超纯水清洗两到三遍，确保其中的乙醇被彻底除去，之后用250ml超纯水重悬；(1) Preparation of column material: 500ml Sepharose Ni 6xFF column material is washed two to three times with ultrapure water to ensure that the ethanol therein is thoroughly removed, and then resuspended with 250ml ultrapure water;
(2)柱管准备：检查所用柱管密封性，润湿柱管底部，静置待用；(2) Preparation of the column tube: check the tightness of the column tube used, wet the bottom of the column tube, and let it stand for use;
(3)平衡：将处理好的柱料沿一侧倒入柱管中，保证柱管垂直于水平面；同时将柱子顶端连接头连接至AKTA avant纯化系统中；插入适配柱并密封；打开系统并调整流速至中流速40ml/min(122.25cm/h)直到柱料不再下降；(3) Balance: Pour the treated column material into the column tube along one side to ensure that the column tube is perpendicular to the horizontal plane; at the same time, connect the top connector of the column to the AKTA avant purification system; insert the adapter column and seal it; open the system And adjust the flow rate to a medium flow rate of 40ml/min (122.25cm/h) until the column material no longer drops;
(4)装柱：等柱料不再下降后，调整流速至高流速80ml/min(244.5cm/h)，运行45min，标记此时柱料位置，下移适配柱至标记位置后再下移3mm，密封；(4) Column loading: After the column material no longer descends, adjust the flow rate to a high flow rate of 80ml/min (244.5cm/h), run for 45 minutes, mark the position of the column material at this time, move the adapter column down to the marked position and then move it down 3mm, sealed;
(5)平衡：用5cv(2500ml)的平衡缓冲液冲洗层析柱，确保系统中A280，254，电导值平衡；所述平衡缓冲液为pH8.0的含有0.5M NaCl、0.02M咪唑的0.05M磷酸钠缓冲液；(5) Equilibration: rinse the chromatography column with 5 cv (2500ml) of equilibration buffer to ensure that A280, 254 and conductance values are balanced in the system; the equilibration buffer is 0.05 pH 8.0 containing 0.5M NaCl, 0.02M imidazole M sodium phosphate buffer;
(6)保存；用1000ml 20％乙醇水溶液冲洗层析柱，之后竖直保存。(6) Preservation; rinse the chromatography column with 1000 ml of 20% ethanol aqueous solution, and then store it vertically.
在本发明中，所述的阳离子交换层析柱料装填方法主要包括以下几个步骤：In the present invention, the described cation exchange chromatography column material packing method mainly includes the following steps:
(1)柱料准备：将500ml SP HP柱料用超纯水清洗两到三遍，确保其中的乙醇被彻底除去，之后用250ml超纯水重悬；(1) Preparation of column material: wash 500ml SP HP column material with ultrapure water two to three times to ensure that the ethanol therein is completely removed, and then resuspend with 250ml ultrapure water;
(2)柱管准备：检查所用柱管密封性，润湿柱管底部，静置待用；(2) Preparation of the column tube: check the tightness of the column tube used, wet the bottom of the column tube, and let it stand for use;
(3)平衡：将处理好的柱料沿一侧倒入柱管中，保证柱管垂直于水平面；同时将柱子顶端连接头连接至纯化系统中；静置20min，插入适配柱并密封；打开系统并调整流速至中流速25ml/min(76.4cm/h)直到柱料不再下降；(3) Balance: Pour the treated column material into the column tube along one side to ensure that the column tube is perpendicular to the horizontal plane; at the same time, connect the top connector of the column to the purification system; let stand for 20 minutes, insert the adapter column and seal; Turn on the system and adjust the flow rate to a medium flow rate of 25ml/min (76.4cm/h) until the column material no longer falls;
(4)装柱：等柱料不再下降后，调整流速至高流速50ml/min(152.8cm/h)，运行45min，标记此时柱料位置，下移适配柱至标记位置后再下移1.5mm，密封；(4) Column loading: After the column material no longer descends, adjust the flow rate to a high flow rate of 50ml/min (152.8cm/h), run for 45 minutes, mark the position of the column material at this time, move the adapter column down to the marked position and then move it down 1.5mm, sealed;
(5)平衡：用5cv(2500ml)的平衡缓冲液冲洗层析柱，确保系统中A280，254，电导值平衡；所述平衡缓冲液为pH8.0的含有0.05M NaCl的0.05M磷酸钠缓冲液；(5) Equilibration: rinse the chromatography column with 5 cv (2500 ml) of equilibration buffer to ensure that A280, 254 and conductance values are balanced in the system; the equilibration buffer is 0.05M sodium phosphate buffer containing 0.05M NaCl at pH 8.0 liquid;
(6)保存；用1000ml 20％乙醇水溶液冲洗层析柱，之后竖直保存。在一些具体实施方案中，(6) Preservation; rinse the chromatography column with 1000 ml of 20% ethanol aqueous solution, and then store it vertically. In some specific embodiments,
本发明中还提供了所述大批量纯化克级力学功能蛋白的层析柱的再生方法。本发明对层析柱的再生方法进行条件筛选，选出适合的再生方法，极大的延长了柱料寿命The invention also provides a method for regenerating the chromatography column for purifying the gram-level mechanically functional protein in large quantities. The invention screens the regeneration method of the chromatography column, selects a suitable regeneration method, and greatly prolongs the life of the column material.
在一些实施方案中，所述大批量纯化克级力学功能蛋白的层析柱的再生方法具体包括如下步骤：In some embodiments, the method for regenerating a chromatography column for bulk purification of gram-level mechanically functional proteins specifically includes the following steps:
(A)将层析柱进行预处理；(A) preprocessing the chromatography column;
(B)用氢氧化钠溶液或含有高浓度氯化钠的氢氧化钠溶液清洗所述预处理后的层析柱，然后用去离子水清洗层析柱，去掉所述层析柱残留清洗液；(B) use sodium hydroxide solution or the sodium hydroxide solution that contains high concentration sodium chloride to wash the chromatography column after described pretreatment, then wash the chromatography column with deionized water, remove the residual cleaning solution of the chromatography column ;
(C)用pH为4.0-5.0的含有TritonX-100的乙二胺四乙酸水溶液清洗所述层析柱；(C) washing the chromatography column with an aqueous solution of EDTA containing TritonX-100 at a pH of 4.0-5.0;
(D)用去离子水清洗层析柱，去掉所述层析柱残留清洗液。(D) Washing the chromatography column with deionized water to remove the residual cleaning solution of the chromatography column.
在本发明中，所述步骤(A)中所述预处理具体为所述镍亲和层析柱用脱镍缓冲液冲洗进行脱镍处理，之后用水清洗掉脱镍缓冲液。所述阳离子交换层析柱仅用水冲洗。In the present invention, the pre-treatment in the step (A) is specifically that the nickel affinity chromatography column is rinsed with a de-nickel buffer solution to perform a de-nickel treatment, and then the de-nickel buffer solution is washed with water. The cation exchange chromatography column was rinsed with water only.
在一些实施方案中，所述脱镍缓冲液为含有50Mm EDTA、PH8.0的20mM磷酸钠缓冲液。In some embodiments, the denickeling buffer is a 20 mM sodium phosphate buffer containing 50 mM EDTA, pH 8.0.
在一些实施方案中，所述镍亲和层析柱用脱镍缓冲液冲洗时所述脱镍缓冲液冲洗的流速为40ml/min。In some embodiments, the flow rate of the nickel-removing buffer flushing when the nickel affinity chromatography column is flushed with the nickel-removing buffer is 40 ml/min.
在本发明中，所述步骤(B)中针对所述镍亲和层析柱用氢氧化钠溶液清洗所述预处理后的层析柱，所述氢氧化钠溶液其浓度为1M。In the present invention, in the step (B), the pretreated column is washed with sodium hydroxide solution for the nickel affinity chromatography column, and the concentration of the sodium hydroxide solution is 1M.
在本发明中，所述步骤(B)中针对所述阳离子交换层析柱用含有高浓度氯化钠的氢氧化钠溶液其为含0.5-2M氯化钠的0.2-1M氢氧化钠溶液。In the present invention, the sodium hydroxide solution containing high concentration sodium chloride is used for the cation exchange chromatography column in the step (B), which is a 0.2-1M sodium hydroxide solution containing 0.5-2M sodium chloride.
在一些实施方案中，所述含有高浓度氯化钠的氢氧化钠溶液其为含2M氯化钠的0.5M氢氧化钠溶液。In some embodiments, the sodium hydroxide solution containing high concentration of sodium chloride is a 0.5M sodium hydroxide solution containing 2M sodium chloride.
在本发明中，所述步骤(C)中所述含有TritonX-100的乙二胺四乙酸水溶液其浓度为0.5-2％体积浓度的TritongX-100和0.1-0.3M的乙二胺四乙酸。In the present invention, the concentration of the EDTA aqueous solution containing TritonX-100 in the step (C) is 0.5-2% volume concentration of TritongX-100 and 0.1-0.3M EDTA.
在一些实施方案中，所述含有TritonX-100的乙二胺四乙酸水溶液其浓度为1％体积浓度的TritongX-100和0.2M的乙二胺四乙酸水溶液。In some embodiments, the aqueous solution of EDTA containing TritonX-100 has a concentration of 1% by volume of TritongX-100 and 0.2M aqueous solution of EDTA.
在一些实施方案中，所述含有TritonX-100的乙二胺四乙酸水溶液其浓度其pH为4.5。In some embodiments, the TritonX-100-containing aqueous EDTA has a concentration of pH 4.5.
在本发明中，对于镍亲和层析柱，在步骤(D)用去离子水清洗层析柱，去掉所述层析柱残留清洗液后还上镍步骤。在一些实施方案中，所述上镍步骤所用溶液为0.1M的硫酸镍缓冲液。In the present invention, for the nickel affinity chromatography column, in step (D), the chromatography column is washed with deionized water, and the nickel step is added after removing the residual washing liquid of the chromatography column. In some embodiments, the solution used in the step of adding nickel is 0.1M nickel sulfate buffer.
在本发明中，所述待纯化样品为ELP融合力学功能蛋白构建pET25b原核表达载体，转化E.coli BLR(DE3)大肠杆菌，经过诱导表达筛选获得的稳定表达菌株发酵获得的菌液收集菌体破碎获得的蛋白。所述与ELP融合的力学功能蛋白包括但不限于丝心蛋白、蛛丝蛋白、节肢弹性蛋白、鱿鱼环齿蛋白。In the present invention, the sample to be purified is an ELP fusion mechanical functional protein to construct a pET25b prokaryotic expression vector, which is transformed into E.coli BLR (DE3) Escherichia coli, and the bacterial liquid obtained by the fermentation of the stable expression strain obtained by inductive expression screening is collected. The protein obtained by crushing. The mechanical functional proteins fused to ELP include, but are not limited to, fibroin, spidroin, elastin, and squid cricodon.
在本发明中，所述柱效的测定方法如下；In the present invention, the method for measuring the column efficiency is as follows;
a)柱平衡：用200ml超纯水清洗柱子，直至A280，254，电导值平衡；a) Column equilibration: wash the column with 200ml ultrapure water until A280, 254, the conductance value is balanced;
b)上样：将200μl柱效测定液加载至柱子中，流速控制在60cm/h；b) Loading:
c)洗脱：用超纯水进行洗脱，流速60cm/h，直至A280，254，电导值平衡；c) Elution: use ultrapure water to elute, the flow rate is 60cm/h, until A280, 254, the conductivity value is balanced;
d)柱效计算：对洗脱峰进行积分，计算出其对称性及每米塔板数；d) Column efficiency calculation: Integrate the elution peak to calculate its symmetry and the number of plates per meter;
e)保存：用200ml 20％乙醇冲洗层析柱，至基线平衡，之后竖直保存。e) Storage: Rinse the column with 200 ml of 20% ethanol until the baseline is equilibrated, and then store vertically.
其中所述柱效测定液为2％质量分数的丙酮水溶液。Wherein, the column efficiency measurement solution is an acetone aqueous solution with a mass fraction of 2%.
本发明的有益效果至少包括下述之一：The beneficial effects of the present invention include at least one of the following:
1)本发明提供了一种节约、快捷可大批量纯化高电荷密度的类弹性蛋白的层析柱的填充方法，经过此方法得到的层析柱结合蛋白量大，损耗小，且再生后杂质残留量大幅度降低，极大的节约了成本并保证蛋白质量，其一次可纯化出克级蛋白，回收率可以达到80％以上且纯度在90％以上；1) The present invention provides a method for filling a chromatography column that is economical, fast and can purify elastin-like proteins with high charge density in large quantities. The chromatography column obtained by this method has a large amount of bound protein, low loss and impurities after regeneration. The residual amount is greatly reduced, which greatly saves the cost and ensures the protein quality. It can purify the gram-level protein at one time, and the recovery rate can reach more than 80% and the purity is more than 90%;
2)由于高电荷密度的类弹性蛋白对柱子的损伤较大且纯化过程中容易拖尾，本发明采用内径较大的柱管，同时填充时使用较高的流速。与通过其他方法装填的层析柱及预装柱相比，每次可以有效减少柱效的损耗至10％以内且有效减少了洗脱峰拖尾现象；2) Since elastin with high charge density causes great damage to the column and is easy to smear during the purification process, the present invention adopts a column tube with a larger inner diameter and uses a higher flow rate during filling. Compared with chromatographic columns and pre-packed columns packed by other methods, the loss of column efficiency can be effectively reduced to less than 10% each time, and the tailing phenomenon of elution peaks can be effectively reduced;
3)通常来说，大体积组装柱均起到初步纯化作用，但回收率及纯度不是十分理想。本发明所述方法得到的层析柱相比于其他大体积层析柱，纯度及回收率均达到了预装柱水准，纯化后得到的蛋白可以用于下一步精细纯化或用于其他领域。3) Generally speaking, large-volume assembled columns play a preliminary purification role, but the recovery rate and purity are not very ideal. Compared with other large-volume chromatography columns, the chromatography column obtained by the method of the present invention has both the purity and the recovery rate reaching the level of the prepacked column, and the purified protein can be used in the next step of fine purification or in other fields.
4)本发明还建立了适用于纯化力学功能蛋白的层析柱的再生方法，所述再生方法针对力学功能蛋白的层析柱再生效果极佳，DNA、蛋白、内毒素、有机碳的残留非常低。4) The present invention also establishes a regeneration method for a chromatography column suitable for purifying mechanically functional proteins. The regeneration method has an excellent regeneration effect on the chromatography column of mechanically functional proteins, and the residues of DNA, protein, endotoxin and organic carbon are very low. Low.
5)本发明所述层析柱的再生方法便捷，所需试剂成本低，可快速去除纯化力学功能蛋白后层析柱上的残留杂质，延长填料寿命，降低使用成本并保证蛋白质量稳定，在短时间内进行下次纯化，提高生产效率。5) The regeneration method of the chromatography column of the present invention is convenient, the cost of the required reagents is low, the residual impurities on the chromatography column after purification of the mechanically functional protein can be quickly removed, the life of the filler is prolonged, the use cost is reduced, and the protein quality is guaranteed to be stable. The next purification can be carried out in a short time to improve production efficiency.
为了进一步理解本发明，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. . Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
如无特殊说明，本发明实施例中所涉及的试剂均为市售产品，均可以通过商业渠道购买获得。Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, which can be purchased through commercial channels.
实施例1Example 1
亲和层析柱所用填料为Ni SepharoseTM6 Fast Flow高流速镍亲和层析填料，填料总镍离子结合量不低于15μmol/ml，线性流速不得高于400cm/h，颗粒尺寸为45-165μm，层析柱结构见图1，其具体填充方式及检测步骤如下：1.亲和层析柱料预处理The packing used in the affinity chromatography column is
(1)取750ml的填料混悬液静置在1000ml量筒中1h，待填料不再下降后倒掉上层液体；(1) Take 750ml of the filler suspension and place it in a 1000ml graduated cylinder for 1 hour, and then pour off the upper layer liquid after the filler no longer descends;
(2)填料中加入500ml超纯水，静置45min，待填料不在下降后倒掉上层液体；(2) Add 500ml of ultrapure water to the filler, let it stand for 45min, and pour off the upper layer liquid when the filler does not drop;
(3)重复步骤(2)三次，直至填料被完全洗净，没有乙醇残留；(3) Repeat step (2) three times until the filler is completely washed, and there is no ethanol residue;
(4)用250ml超纯水重悬填料，准备装柱。(4) Resuspend the filler with 250ml ultrapure water, and prepare to pack the column.
2.层析柱装填2. Column packing
2.1溶液配制2.1 Solution preparation
平衡缓冲液：Equilibration buffer:
2.2层析柱装填2.2 Column packing
(1)柱管准备：检查所用内径50mm柱管，确保其密封性良好，润湿柱管底部，静置待用；(1) Preparation of column tube: Check the column tube with an inner diameter of 50mm to ensure that it has good sealing performance, wet the bottom of the column tube, and let it stand for use;
(2)平衡：将处理好的柱料沿一侧倒入柱管中，保证柱管垂直于水平面；同时将柱子顶端连接头连接至纯化系统中；插入适配柱并密封；将系统A泵接入到抽滤过的超纯水中，打开系统并调整流速至40ml/min(122.25cm/h)；(2) Balance: Pour the treated column material into the column tube along one side to ensure that the column tube is perpendicular to the horizontal plane; at the same time, connect the top connector of the column to the purification system; insert the adapter column and seal it; pump the system A Access to the ultrapure water filtered by suction, turn on the system and adjust the flow rate to 40ml/min (122.25cm/h);
(3)装柱：等柱料不再下降后，打开系统并调整流速至80ml/min(244.5cm/h)，运行45min，标记此时柱料位置，柱长25.5cm，下移适配柱至标记位置后再下移3mm，密封；(3) Column loading: After the column material no longer descends, turn on the system and adjust the flow rate to 80ml/min (244.5cm/h), run for 45min, mark the position of the column material at this time, the column length is 25.5cm, and move down to adapt the column Move down 3mm to the marked position and seal;
(4)平衡：用2500ml的平衡缓冲液冲洗层析柱，最终系统中A280，254，电导值平衡；(4) Equilibrium: rinse the chromatography column with 2500ml of equilibration buffer, and in the final system, A280, 254, and conductance value balance;
(5)清洗及保存；用1000ml 20％乙醇冲洗层析柱，之后竖直保存。(5) Washing and storage; the column was washed with 1000 ml of 20% ethanol, and then stored vertically.
3.柱效损耗测定3. Determination of column efficiency loss
3.1溶液配制3.1 Solution preparation
柱效测定液Column Efficiency Assay Solution
2％质量分数的丙酮水溶液 50mL2% mass fraction of acetone aqueous solution 50mL
丙酮 1gAcetone 1g
超纯水 49gUltrapure Water 49g
3.2柱效测定：3.2 Determination of column efficiency:
柱效测定需要在每轮纯化之前及之后进行，柱效损耗＝纯化前塔板数(N/m)/纯化后塔板数(N/m)×100％，测定方法如下：The column efficiency measurement needs to be carried out before and after each round of purification. The column efficiency loss = the number of plates before purification (N/m) / the number of plates after purification (N/m) × 100%. The measurement method is as follows:
(1)柱平衡：用2000ml超纯水清洗柱子，直至A280、254，电导值平衡；(1) Column balance: wash the column with 2000ml ultrapure water until A280, 254, the conductance value is balanced;
(2)上样：将200μl柱效测定液加载至柱子中，流速控制在60cm/h；(2) Loading:
(3)洗脱：用超纯水进行洗脱，流速60cm/h，直至A280、254，电导值平衡；(3) Elution: use ultrapure water to elute, the flow rate is 60cm/h, until A280, 254, the conductivity value is balanced;
(4)柱效计算：对洗脱峰进行积分，计算出其对称性及每米塔板数，纯化前塔板数为18902.8、纯化后为17390损耗为8％；(4) Column efficiency calculation: Integrate the elution peak, calculate its symmetry and the number of plates per meter, the number of plates before purification is 18902.8, after purification it is 17390 and the loss is 8%;
(5)清洁及保存：用2000ml 20％乙醇冲洗层析柱，至基线平衡，之后竖直保存。(5) Cleaning and storage: Rinse the column with 2000 ml of 20% ethanol until the baseline is equilibrated, and then store it vertically.
4.力学功能蛋白的亲和层析4. Affinity chromatography of mechanically functional proteins
将高压破碎处理后的力学功能蛋白通过此层析柱进行纯化，具体纯化步骤为：The mechanically functional protein after high pressure crushing treatment is purified by this chromatography column, and the specific purification steps are:
4.1缓冲液配制4.1 Buffer preparation
(1)平衡缓冲液：(1) Equilibration buffer:
加Hcl调整pH8.0Add HCl to adjust pH to 8.0
(2)洗脱缓冲液：(2) Elution buffer:
用平衡缓冲液配制成咪唑终浓度为0.35M的缓冲液作为洗脱缓冲液，pH调整为8.0；The equilibration buffer was used to prepare a buffer with a final imidazole concentration of 0.35M as the elution buffer, and the pH was adjusted to 8.0;
(3)洗柱缓冲液：(3) Column washing buffer:
用平衡缓冲液配置成含1M NaCl的缓冲液作为洗柱缓冲液，pH8.0；Use equilibration buffer to configure the buffer containing 1M NaCl as the column washing buffer, pH 8.0;
(4)清洗液：(4) Cleaning solution:
20％乙醇水溶液用于镍柱的清洁及保存，但在必要情况下，需要对镍柱进行脱镍再生处理。The 20% ethanol aqueous solution is used for the cleaning and preservation of the nickel column, but if necessary, the nickel column needs to be de-nickeled and regenerated.
4.2力学功能蛋白镍柱纯化步骤：4.2 Purification steps of mechanical functional protein nickel column:
(1)柱平衡：用20％乙醇水溶液洗涤Ni柱，之后用平衡缓冲液进行平衡，应冲洗到A280，A254基线平衡，电导及pH稳定时，即所有的未被结合的分子都被冲出镍柱。(1) Column equilibration: Wash the Ni column with 20% ethanol aqueous solution, and then equilibrate with equilibration buffer. It should be washed to A280, A254 baseline equilibrium, when the conductance and pH are stable, that is, all unbound molecules are flushed out Nickel column.
(2)上样：将处理好的含有力学功能蛋白的样品加载到平衡后的Ni柱中，流速控制在15ml/min。(2) Loading: Load the processed sample containing the mechanically functional protein into the equilibrated Ni column, and control the flow rate at 15 ml/min.
(3)平衡：用2500ml的平衡缓冲液继续冲洗Ni柱，流速为40ml/min，使未结合的组分从柱子上分离，冲洗至A280，A254基线平衡，电导及pH稳定。(3) Equilibration: Continue to rinse the Ni column with 2500ml of equilibration buffer at a flow rate of 40ml/min to separate unbound components from the column, rinse to A280, A254 baseline balance, conductance and pH are stable.
(4)洗脱：用总计5000ml的含有0-100％洗脱缓冲液的平衡缓冲液进行洗脱，流速为40ml/min，使结合后的目的蛋白被充分洗脱下来，当系统显示A280，A254基线开始上升时收集洗脱液并保存，纯化过程中UV280变化峰见图4及图6。(4) Elution: Elute with a total of 5000ml of equilibration buffer containing 0-100% elution buffer at a flow rate of 40ml/min, so that the bound target protein is fully eluted. When the system displays A280, When the A254 baseline began to rise, the eluate was collected and stored. The UV280 change peak during the purification process is shown in Figure 4 and Figure 6.
(5)镍柱的清洁及保存：用洗柱缓冲液对镍柱进行彻底的清洁，保证系统中所显示的电导值为零且A280及A254平衡为止，将清洗并用乙醇填充好的柱子进行柱效测定。(5) Cleaning and storage of the nickel column: Thoroughly clean the nickel column with the column washing buffer to ensure that the conductivity value displayed in the system is zero and the A280 and A254 are balanced. Efficacy determination.
(6)纯化后蛋白保存：其纯度测定依据《中国药典2015版》通则0541第五法所提供的检测方式。将待测样品进行SDS-聚丙烯酰胺凝胶电泳，染色后用凝胶扫描仪进行扫描，对目的蛋白条带对应峰面积进行归一化处理并与其他杂蛋白条带进行比较，得到相应纯度，为95％。实验结果见图2。放入到-80℃冰箱中保存以便进行下一步实验。(6) Preservation of protein after purification: its purity is determined according to the detection method provided by the fifth method of general rule 0541 of "Chinese Pharmacopoeia 2015 Edition". The samples to be tested were subjected to SDS-polyacrylamide gel electrophoresis, scanned with a gel scanner after staining, and the corresponding peak areas of the target protein bands were normalized and compared with other impurity protein bands to obtain the corresponding purity. , is 95%. The experimental results are shown in Figure 2. Stored in a -80°C freezer for further experiments.
5.力学功能蛋白亲和层析柱的再生5. Regeneration of Mechanofunctional Protein Affinity Chromatography Columns
对使用完毕的亲和层析柱进行再生，其步骤如下：The steps of regenerating the used affinity chromatography column are as follows:
(1)将纯化后的镍亲和层析柱(填料型号为Ni Sepharose FF，柱管为GE XK 50/30，高30cm，直径5cm，柱体积为500ml)用2000ml 50Mm EDTA、pH8.0的20mM磷酸钠缓冲液冲洗，流速为40ml/min，之后用2000ml水清洗层析柱，去除掉脱镍缓冲液；(1) The purified nickel affinity chromatography column (filler model is Ni Sepharose FF, column tube is
(2)用2000ml含有1M氢氧化钠溶液清洗所述层析柱，然后用2000ml去离子水清洗层析柱，去掉层析柱残留清洗液；(2) cleaning the chromatography column with 2000ml containing 1M sodium hydroxide solution, then cleaning the chromatography column with 2000ml deionized water, removing the residual cleaning solution of the chromatography column;
(3)用1000ml含有1％TritonX-100的pH4.5的0.2M乙二胺四乙酸水溶液清洗所述层析柱；(3) washing the chromatography column with 1000ml of 0.2M ethylenediaminetetraacetic acid aqueous solution at pH 4.5 containing 1% TritonX-100;
(4)用2000ml去离子水清洗层析柱，去掉层析柱残留清洗液，之后用200ml 0.1 M NiSO4进行上镍步骤，完成清洗。(4) Wash the chromatography column with 2000 ml of deionized water, remove the residual cleaning solution of the chromatography column, and then use 200 ml of 0.1 M NiSO 4 to carry out the step of adding nickel to complete the cleaning.
(5)取步骤(4)去离子水清洗后得到的清洗液，采用DIG-DNA及BCA法检测其残留的DNA及蛋白量，分别为20pg/ml和100ng/ml，并用总有机碳分析仪分析其有机碳含量为120ppb，低于500ppb，用内毒素分析仪分析后内毒素含量低于0.04 EU/ml，说明清洗合格，结果见表1。(5) Take the cleaning solution obtained after washing with deionized water in step (4), use DIG-DNA and BCA methods to detect the amount of residual DNA and protein, which are 20pg/ml and 100ng/ml respectively, and use a total organic carbon analyzer Analysis of its organic carbon content is 120ppb, lower than 500ppb, and the endotoxin content is lower than 0.04 EU/ml after analysis with an endotoxin analyzer, indicating that the cleaning is qualified, and the results are shown in Table 1.
实施例2-5和对比例1-2与实施例1的方法相同，不同之处为氯化钠，氢氧化钠及TritonX-100的浓度不同，详情见表1。Examples 2-5 and Comparative Examples 1-2 are the same as those of Example 1, except that the concentrations of sodium chloride, sodium hydroxide and TritonX-100 are different, see Table 1 for details.
表1实施例1-5及对比例1-2清洗结果对比Table 1 embodiment 1-5 and comparative example 1-2 cleaning result comparison
通过表1可知，采用实施例1-5的清洗方法，清洗后清洗液的各项残留指标均达到了预期标准。而对比例1及2中，清洗液采用较低浓度时，层析柱残留杂质不合格。As can be seen from Table 1, using the cleaning methods of Examples 1-5, the residual indicators of the cleaning solution after cleaning have all reached the expected standards. In Comparative Examples 1 and 2, when the cleaning solution adopts a lower concentration, the residual impurities in the chromatographic column are unqualified.
实施例6-7、对比例3与实施例1的方法相同，不同之处为乙二胺四乙酸和TritonX-100混合液中乙二胺四乙酸的浓度不同，详情见表2。Examples 6-7 and Comparative Example 3 are the same as those of Example 1, except that the concentrations of EDTA in the mixed solution of EDTA and TritonX-100 are different, see Table 2 for details.
表2实施例6-7及对比例3清洗结果对比Table 2 embodiment 6-7 and comparative example 3 cleaning result comparison
通过表2可知，本发明中采用0.2M-0.3M的清洗液清洗后层析柱的残留DNA及蛋白含量较低，并且内毒素及总有机碳均达到了相应指标，对比例3中采用了较低浓度乙二胺四乙酸，内毒素及有机碳均超标，且残留DNA和蛋白浓度极高，清洗不合格，因此，本发明采用的乙二胺四乙酸浓度为0.2M。As can be seen from Table 2, in the present invention, the residual DNA and protein content of the chromatographic column after washing with a 0.2M-0.3M cleaning solution are relatively low, and both endotoxin and total organic carbon have reached the corresponding indicators. At lower concentrations of EDTA, both endotoxin and organic carbon exceed the standard, and the residual DNA and protein concentrations are extremely high, and the cleaning is unqualified. Therefore, the concentration of EDTA used in the present invention is 0.2M.
实施例8Example 8
阳离子交换层析柱所用填料为SP Sepharose High Performance高分辨率阳离子交换层析填料，填料氢离子结合量不低于0.15到0.20mmol/ml，线性流速不得高于150cm/h，颗粒尺寸为34μm，层析柱具体填充方法及检测步骤如下：The packing used in the cation exchange chromatography column is SP Sepharose High Performance high-resolution cation exchange chromatography packing. The specific packing method and detection steps of the chromatography column are as follows:
1.阳离子交换层析柱料预处理1. Cation exchange chromatography column pretreatment
(1)取750ml的填料混悬液静置在1000ml量筒中1h，待填料不再下降后倒掉上层液体；(1) Take 750ml of the filler suspension and place it in a 1000ml graduated cylinder for 1 hour, and then pour off the upper layer liquid after the filler no longer descends;
(2)填料中加入500ml超纯水，静置45min，待填料不在下降后倒掉上层液体；(2) Add 500ml of ultrapure water to the filler, let it stand for 45min, and pour off the upper layer liquid when the filler does not drop;
(3)重复步骤(2)三次，直至填料被完全洗净，没有乙醇残留；(3) Repeat step (2) three times until the filler is completely washed, and there is no ethanol residue;
(4)用250ml超纯水重悬填料，准备装柱。(4) Resuspend the filler with 250ml ultrapure water, and prepare to pack the column.
2.层析柱装填2. Column packing
2.1溶液配制2.1 Solution preparation
平衡缓冲液：Equilibration buffer:
pH＝8.0的磷酸钠缓冲液，添加氯化钠 5LSodium phosphate buffer pH=8.0, add sodium chloride 5L
磷酸钠(M＝163.94) 81.97gSodium Phosphate (M=163.94) 81.97g
氯化钠(M＝58.44) 14.61gSodium chloride (M=58.44) 14.61g
2.2层析柱装填2.2 Column packing
(1)柱管准备：检查所用内径50mm柱管，确保其密封性良好，润湿柱管底部，静置待用；(1) Preparation of column tube: Check the column tube with an inner diameter of 50mm to ensure that it has good sealing performance, wet the bottom of the column tube, and let it stand for use;
(2)平衡：将处理好的柱料沿一侧倒入柱管中，保证柱管垂直于水平面；同时将柱子顶端连接头连接至纯化系统中；插入适配柱并密封；静置20min，之后将系统A泵接入到抽滤过的超纯水中，打开系统并调整流速至25ml/min(76.40cm/h)；(2) Balance: Pour the treated column material into the column tube along one side to ensure that the column tube is perpendicular to the horizontal plane; at the same time, connect the top connector of the column to the purification system; insert the adapter column and seal it; let it stand for 20 minutes, Then connect the system A pump to the ultrapure water filtered by suction, turn on the system and adjust the flow rate to 25ml/min (76.40cm/h);
(3)装柱：等柱料不再下降后，打开系统并调整流速至50ml/min(152.8cm/h)，运行45min，标记此时柱料位置，柱长25.3cm，下移适配柱至标记位置后再下移1.5mm，密封；(3) Column loading: After the column material no longer descends, turn on the system and adjust the flow rate to 50ml/min (152.8cm/h), run for 45min, mark the position of the column material at this time, the column length is 25.3cm, and move down to adapt the column Move down 1.5mm to the marked position and seal;
(4)平衡：用2500ml的平衡缓冲液冲洗层析柱，最终系统中A280，254，电导值平衡；(4) Equilibrium: rinse the chromatography column with 2500ml of equilibration buffer, and in the final system, A280, 254, and conductance value balance;
(5)清洗及保存；用1000ml 20％乙醇冲洗层析柱，之后竖直保存。(5) Washing and storage; the column was washed with 1000 ml of 20% ethanol, and then stored vertically.
3.柱效损耗测定3. Determination of column efficiency loss
3.1溶液配制3.1 Solution preparation
柱效测定液Column Efficiency Assay Solution
2％质量分数的丙酮水溶液 50mL2% mass fraction of acetone aqueous solution 50mL
丙酮 1gAcetone 1g
超纯水 49gUltrapure Water 49g
3.2柱效测定：3.2 Determination of column efficiency:
柱效测定需要在每轮纯化之前及之后进行，柱效损耗＝纯化前塔板数(N/m)/纯化后塔板数(N/m)×100％，测定方法如下The column efficiency measurement needs to be carried out before and after each round of purification. The column efficiency loss = the number of plates before purification (N/m) / the number of plates after purification (N/m) × 100%, the measurement method is as follows
(1)柱平衡：用2000ml超纯水清洗柱子，直至A280，254，电导值平衡；(1) Column balance: wash the column with 2000ml ultrapure water until A280, 254, the conductance value is balanced;
(2)上样：将200μl柱效测定液加载至柱子中，流速控制在60cm/h；(2) Loading:
(3)洗脱：用超纯水进行洗脱，流速60cm/h，直至A280，254，电导值平衡；(3) Elution: use ultrapure water for elution, flow rate 60cm/h, until A280, 254, the conductivity value is balanced;
(4)柱效计算：对洗脱峰进行积分，计算出其对称性及每米塔板数，纯化前塔板数为30521、纯化后为28995损耗为5％；(4) Column efficiency calculation: Integrate the elution peak, calculate its symmetry and the number of plates per meter, the number of plates before purification is 30521, after purification it is 28995 and the loss is 5%;
(5)清洁及保存：用2000ml 20％乙醇冲洗层析柱，至基线平衡，之后竖直保存。(5) Cleaning and storage: Rinse the column with 2000 ml of 20% ethanol until the baseline is equilibrated, and then store it vertically.
4.力学功能蛋白的离子交换层析4. Ion-Exchange Chromatography of Mechanically Functional Proteins
将高压破碎处理后的力学功能蛋白通过此层析柱进行纯化，具体纯化步骤为：The mechanically functional protein after high pressure crushing treatment is purified by this chromatography column, and the specific purification steps are:
4.1缓冲液配制4.1 Buffer preparation
(1)平衡缓冲液：(1) Equilibration buffer:
pH＝8.0的磷酸钠缓冲液，添加氯化钠 5LSodium phosphate buffer pH=8.0, add sodium chloride 5L
磷酸钠(M＝163.94) 81.97gSodium Phosphate (M=163.94) 81.97g
氯化钠(M＝58.44) 14.91gSodium chloride (M=58.44) 14.91g
加Hcl调整pH＝8.0Add HCl to adjust pH=8.0
(2)洗脱缓冲液：(2) Elution buffer:
用平衡缓冲液A配制成氯化钠终浓度为2M的缓冲液作为洗脱缓冲液，pH调整为8.0Equilibration buffer A was used to prepare a buffer with a final concentration of 2M sodium chloride as the elution buffer, and the pH was adjusted to 8.0
(3)洗柱缓冲液：(3) Column washing buffer:
含有2M氯化钠的氢氧化钠溶液 5LSodium hydroxide solution containing 2M sodium chloride 5L
氢氧化钠(M＝40) 100gSodium hydroxide (M=40) 100g
氯化钠(M＝58.44) 584.4gSodium chloride (M=58.44) 584.4g
(4)清洗液：20％乙醇用于离子交换柱的清洁及保存。(4) Cleaning solution: 20% ethanol is used for cleaning and preservation of the ion exchange column.
4.2力学功能蛋白SP柱纯化步骤：4.2 Purification steps of mechanical functional protein SP column:
(1)柱平衡：用20％乙醇水溶液洗涤离子交换柱，之后用平衡缓冲液进行平衡，应冲洗到A280，A254基线平衡，电导及pH稳定时，即所有的未被结合的分子都被冲出离子交换柱。(1) Column equilibration: Wash the ion exchange column with 20% ethanol aqueous solution, and then equilibrate with equilibration buffer. It should be washed to A280, A254 baseline equilibrium, when the conductance and pH are stable, that is, all unbound molecules are washed. out of the ion exchange column.
(2)上样：将处理好的含有力学功能蛋白的样品加载到平衡后的离子交换柱中，流速控制在10ml/min。(2) Loading: Load the processed sample containing the mechanically functional protein into the equilibrated ion exchange column, and control the flow rate at 10 ml/min.
(3)平衡：用2500ml的平衡缓冲液继续冲洗离子交换柱，流速为30ml/min，使未结合的组分从柱子上分离，冲洗至A280，A254基线平衡，电导及pH稳定。(3) Equilibration: Continue to rinse the ion exchange column with 2500ml of equilibration buffer at a flow rate of 30ml/min to separate unbound components from the column, rinse to A280, A254 baseline balance, conductance and pH are stable.
(4)洗脱：用总计5000ml的含有0-100％洗脱缓冲液的平衡缓冲液进行洗脱，流速为30ml/min，使结合后的目的蛋白被充分洗脱下来，当系统显示A280，A254基线开始上升时收集洗脱液并保存。纯化过程中UV280变化峰见图5及图7。(4) Elution: Elute with a total of 5000ml of equilibration buffer containing 0-100% elution buffer at a flow rate of 30ml/min, so that the bound target protein is fully eluted. When the system displays A280, The eluate was collected and stored when the A254 baseline began to rise. The UV280 change peaks during the purification process are shown in Figures 5 and 7.
(5)离子交换柱的再生及保存：用2000ml洗柱缓冲液对离子交换柱进行彻底的清洁，流速为30ml/min，保证系统中所显示的电导值为零且A280及A254平衡为止，将清洗并用乙醇填充好的柱子进行柱效测定。(5) Regeneration and preservation of the ion exchange column: Thoroughly clean the ion exchange column with 2000ml of column washing buffer, the flow rate is 30ml/min, and ensure that the conductance value displayed in the system is zero and A280 and A254 are in equilibrium. The column was cleaned and packed with ethanol for efficiency determination.
(6)纯化后蛋白保存，将纯化后的蛋白进行纯度测定，其纯度测定依据《中国药典2015版》通则0541第五法所提供的检测方式。将待测样品进行SDS-聚丙烯酰胺凝胶电泳，染色后用凝胶扫描仪进行扫描，对目的蛋白条带对应峰面积进行归一化处理并与其他杂蛋白条带进行比较，得到相应纯度，为96％。实验结果见图3。放入到-80℃冰箱中保存以便进行下一步实验。(6) The purified protein is stored, and the purified protein is subjected to purity determination, and its purity determination is based on the detection method provided by the fifth method of the general rule 0541 of the "Chinese Pharmacopoeia 2015 Edition". The samples to be tested were subjected to SDS-polyacrylamide gel electrophoresis, scanned with a gel scanner after staining, and the corresponding peak areas of the target protein bands were normalized and compared with other impurity protein bands to obtain the corresponding purity. , was 96%. The experimental results are shown in Figure 3. Stored in a -80°C freezer for further experiments.
5.力学功能蛋白离子交换层析柱的再生5. Regeneration of Mechanically Functional Protein Ion-Exchange Chromatography Columns
对使用完毕的离子交换层析柱进行再生，其步骤如下：To regenerate the used ion exchange chromatography column, the steps are as follows:
(1)将纯化使用后的离子交换层析柱(填料型号为SP Sepharose HP，柱管为GE XK 50/30，高30cm，直径5cm，柱体积为500ml)用2000ml水清洗层析柱，去除掉20％乙醇；(1) The ion-exchange chromatography column (filler model is SP Sepharose HP, column tube is
(2)用2000ml含有2M氯化钠的0.5M氢氧化钠溶液清洗所述层析柱，然后用2000ml去离子水清洗层析柱，去掉层析柱残留清洗液；(2) cleaning the chromatography column with 2000ml of 0.5M sodium hydroxide solution containing 2M sodium chloride, then cleaning the chromatography column with 2000ml deionized water, removing the residual cleaning solution of the chromatography column;
(3)取步骤(2)去离子水清洗后得到的清洗液，采用DIG-DNA及BCA法检测其残留的DNA及蛋白量，分别为20pg/ml和100ng/ml，并用总有机碳分析仪分析其有机碳含量为120ppb，低于500ppb，用内毒素分析仪分析后内毒素含量低于0.04EU/ml，说明清洗合格。(3) Take the cleaning solution obtained after washing with deionized water in step (2), use DIG-DNA and BCA methods to detect the amount of residual DNA and protein, which are 20pg/ml and 100ng/ml respectively, and use a total organic carbon analyzer Analysis of its organic carbon content is 120ppb, less than 500ppb, and the endotoxin content is less than 0.04EU/ml after analysis with an endotoxin analyzer, indicating that the cleaning is qualified.
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