KR102527221B1 - A method for purification of target protein - Google Patents

Abstract

The present invention provides a method for purifying proteins.

Description

Protein purification method {A method for purification of target protein}

The present invention relates to a method for purifying proteins.

The new coronavirus (2019-nCoV) that occurred in Wuhan, China in 2019 was named COVID-19 in the sense that human infection was first confirmed in the second half of 2019, and it is a virus that causes respiratory diseases in humans and animals. It is known that various symptoms appear after an incubation period of up to two weeks at work. The main symptoms include lethargy, high fever of 37.5 degrees or higher, cough, sore throat, phlegm, muscle pain, headache, shortness of breath and pneumonia, and respiratory failure due to lung damage. Similar diseases include MERS and SARS, which have a lower mortality rate than MERS or SARS, but are known to be much more contagious. There is an increasing demand in the medical industry for the production of efficient vaccines for the prevention of COVID-19 and the production of vaccine antigen proteins.

Accordingly, the present inventors developed a method for producing and purifying proteins used in coronavirus vaccines, and completed the present invention.

US 2021-0338807 A1

An object of the present invention is to provide a method for purifying a target protein, comprising the steps of performing (i) primary anion exchange chromatography and (ii) secondary anion exchange chromatography using a solution containing the target protein. will be.

One aspect of the present invention is a method for purifying a protein of interest.

In one embodiment, it relates to a method for purifying a target protein comprising the steps of performing (i) first anion exchange chromatography and (ii) second anion exchange chromatography using a solution containing the target protein. .

As the purification method according to the preceding embodiment, the target protein is SEQ ID NO: 1 or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 It relates to a method for purifying a target protein having an identity of %, 98% or 99% or more.

As the purification method according to any one of the above embodiments, the target protein is recovered from a strain expressing the protein, it relates to a purification method of the target protein.

The purification method according to any one of the above embodiments, wherein the target protein is recovered in the form of an inclusion body.

The purification method according to any one of the preceding embodiments, wherein the primary anion exchange chromatography comprises the step of loading an anion exchange resin column filled with a resin having a secondary or tertiary amine as an exchange group Purification of a target protein It's about how.

As the purification method according to any one of the preceding embodiments, the primary anion exchange chromatography is PEI (Polyethyleneimine), PAB (Para-aminobenzyl), AE (amino ethyl), DEAE (diethyl aminoethyl) or DMAE (dimethyl aminoethyl) column It relates to a method for purifying a target protein, which is to use.

As the purification method according to any one of the above embodiments, the first anion exchange chromatography relates to a method for purifying a target protein, including performing in a negative mode.

As the purification method according to any one of the above embodiments, the secondary anion exchange chromatography relates to a method for purifying a target protein, including performing in a positive mode.

As a purification method according to any one of the preceding embodiments, the first anion exchange chromatography is performed in a negative mode and then the second anion exchange chromatography is performed in a positive mode, It relates to a method for purifying a target protein.

The purification method according to any one of the preceding embodiments, wherein the secondary anion exchange chromatography comprises the step of loading an anion exchange resin column filled with a resin having a secondary or tertiary amine as an exchange group Purification of a target protein It's about how.

As a purification method according to any one of the preceding embodiments, the secondary anion exchange chromatography is PEI (Polyethyleneimine), PAB (Para-aminobenzyl), AE (amino ethyl), DEAE (diethyl aminoethyl) or DMAE (dimethyl aminoethyl) column It relates to a method for purifying a target protein, which is to use.

As the purification method according to any one of the above embodiments, the buffer of the secondary anion exchange chromatography relates to a method for purifying a target protein including a detergent.

As the purification method according to any one of the above embodiments, the content of the detergent is 0.01 to 5% (v / v), it relates to a purification method of a target protein.

As the purification method according to any one of the preceding embodiments, the secondary anion exchange chromatography comprises first washing the resin using a buffer containing detergent and salt; It relates to a method for purifying a target protein, comprising the step of secondarily washing a resin using a buffer containing detergent and salt.

As the purification method according to any one of the above embodiments, the buffer in the washing step of the secondary anion exchange chromatography is a phosphate buffer containing salt, and relates to a method for purifying a target protein.

As the purification method according to any one of the above embodiments, the salt is sodium chloride, and relates to a method for purifying a target protein.

As the purification method according to any one of the preceding embodiments, the sodium chloride is contained in the buffer of the second washing step at a concentration of 150 mM or more and less than 250 mM, and relates to a method for purifying a target protein.

As the purification method according to any one of the above embodiments, the detergent included in the secondary anion exchange chromatography buffer is Triton X-100, and relates to a method for purifying a target protein.

As the purification method according to any one of the above embodiments, the purification method relates to a method for purifying a target protein, further comprising filtering a solution recovered from secondary chromatography.

As the purification method according to any one of the preceding embodiments, the filtration relates to a method for purifying a target protein, which is ultrafiltration and diafiltration.

As the purification method according to any one of the preceding embodiments, the buffer of the ultrafiltration process relates to a method for purifying a target protein comprising Tris, sodium chloride and a detergent.

As the purification method according to any one of the preceding embodiments, the detergent included in the buffer of the ultrafiltration step is CHAPS, and relates to a method for purifying a target protein.

As the purification method according to any one of the above embodiments, the content of the detergent contained in the buffer solution of the ultrafiltration step is 0.01% to 5% (v / v), it relates to a method for purifying a target protein.

As the purification method according to any one of the above embodiments, the purification method relates to a method for purifying a target protein without performing an additional chromatography step.

As the purification method according to any one of the preceding embodiments, the purification method uses a solution containing SEQ ID NO: 1 or a target protein having 75% or more identity thereto, (i) having a secondary or tertiary amine as an exchange group performing first anion exchange chromatography in negative mode using a column filled with resin; (ii) performing secondary anion exchange chromatography in a positive mode using a column packed with a resin having a secondary or tertiary amine as an exchange group and an equilibration, washing and elution buffer containing a detergent; and (iii) performing ultrafiltration and diafiltration in the presence of a buffer containing a detergent.

The purification method according to any one of the preceding embodiments, wherein the detergent is included in an amount of 0.01 to 5% (v / v), (iii) the detergent contained in the ultrafiltration and diafiltration buffer is CHAPS, purification of the target protein It's about how.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below. In addition, a number of papers and patent documents are referenced throughout this specification and their citations are indicated. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention belongs and the contents of the present invention.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be included in this invention.

As used herein, the term "about" may be preceded by a specific numerical value. As used in this application, the term "about" includes not only the exact number that follows the term, but also a range that is or is close to that number. It can be determined whether the number is close to or nearly the specific number mentioned, given the context in which it is presented. As an example, the term “about” can refer to a range of -10% to +10% of a numerical value. As another example, the term "about" can refer to a range of -5% to +5% of a given numerical value. However, it is not limited thereto.

As used herein, the term “and/or” when used herein is to be understood as specifically disclosing each of the two specified features or elements, either with or without the other. Thus, the term "and/or" as used herein in phrases such as "A and/or B" includes "A and B", "A or B", "A" (alone), and "B" (alone). It is intended to include Likewise, the term "and/or" as used in phrases such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the term "consisting essentially of" means that a non-specified ingredient may be present if the characteristics of the claimed subject matter are not substantially affected by the presence of the non-specified ingredient.

As used herein, the term “consisting of” means that the proportions of the specified component(s) total 100%. Ingredients or features that come after the term “consisting of” may be essential or mandatory. In some embodiments, in addition to the ingredients or features that come under "consisting of", any other ingredients or non-essential ingredients may be excluded.

As used herein, the term "comprising" refers to the presence of any of the features, steps or components described below the term, and does not exclude the presence or addition of one or more features, steps or components. Components or features described herein under "comprising" may be required or mandatory, but in some embodiments may further include other optional or non-essential components or features.

As used herein, the term “comprising” may, in some embodiments, be modified to refer to “consisting essentially of” or “consisting of”.

With reference to amino acid sequences herein, a polypeptide "comprising" the amino acid sequence set forth in a particular SEQ ID NO, a polypeptide "consisting of' the amino acid sequence set forth in a particular SEQ ID NO, or a polypeptide or protein that "has" the amino acid sequence set forth in a particular SEQ ID NO. Even if it is described, as long as it has the same or equivalent activity as the polypeptide consisting of the amino acid sequence of the corresponding sequence number, proteins having amino acid sequences in which some sequences have been deleted, modified, substituted, conservatively substituted or added can also be used in this application. It is self-evident that it can For example, if the amino acid sequence has an addition of a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation thereof, or a conservative substitution to the N-terminus and/or C-terminus of the amino acid sequence. It may, but is not limited thereto.

One aspect of the present invention is a method for purifying a target protein, comprising the steps of performing (i) primary anion exchange chromatography and (ii) secondary anion exchange chromatography using a solution containing the target protein. to provide.

The "protein of interest" can be used to prepare multimeric protein assemblies that display antigens or antigenic fragments for use in the preparation of immunogenic compositions. The target protein may be a protein disclosed in WO2019-169120, US 9630994 B2. Specifically, the target protein of the present invention may be the protein represented by SEQ ID NO: 1, but those having the same function are included without limitation.

In one embodiment, the protein of interest of the present invention may contain any modification that does not disrupt assembly into a multimeric or multimeric protein assembly, including, for example, addition, deletion, insertion, substitution or modification of amino acids. can do. Examples include cases involving conservative sequence modifications.

In the present invention, "conservative sequence modifications" may refer to amino acid modifications that do not significantly affect or change the structural properties of a protein of interest. Such conservative modifications include amino acid substitutions, additions and deletions. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Such substitutions are generally based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Families of amino acid residues with similar side chains have been defined in the art. This family includes basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), non-charged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. For example, tyrosine, phenylalanine, tryptophan, histidine).

In one embodiment, the target protein of the present invention is SEQ ID NO: 1 or at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, It may have sequences with greater than 98% or 99% identity. In any one of the above embodiments, the target protein of the present invention comprises or consists essentially of the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, 7, 8, 9, 10 or 11 It may consist essentially of or consist of. In any one of the foregoing embodiments, the protein of interest of the invention has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 identified interfaces ) contains the same amino acid sequence at the position. In the target protein, the interface residues identified as being present at the interface to be assembled to form the nanostructure are 24, 28, 36, 124, 125, 127, 128, 129, 131 from the N-terminus of SEQ ID NO: 1. , 132, 133, 135, and 139. The protein of SEQ ID NO: 1 herein may be referred to as "Component B". A plurality of isolated protein monomers of SEQ ID NO: 1 may self-assemble through interactions to form a pentamer. This assembly can be achieved through non-covalent protein-protein interaction reactions.

Such Component B can self-assemble with other proteins to form an immunogenic composition, and for the purpose of the present application, it may be one component of a vaccine against coronavirus. As raw materials for mass production of such vaccines, high purity and/or mass production and/or purification of Component B is required, and the present invention provides a purification method capable of mass-producing Component B with high purity.

The purification method of the present invention is characterized by performing first anion exchange chromatography and second anion exchange chromatography.

In the present invention, "anion exchange chromatography (AEX)" can separate molecules according to their charge by binding negatively charged (or acidic) molecules to a positively charged support, Homologues of molecules (acidic, basic and neutral) can be easily separated by this technique.

An “anion exchange resin” or “anion exchange membrane” used in anion exchange chromatography refers to a solid phase that is positively charged and thus has one or more positively charged ligands attached thereto. Any positively charged ligand attached to a solid phase suitable for forming an anion exchange resin may be used.

Examples of commercially available anion exchange resins are Poros HQ, Poros XQ, Q Sepharose Fast Flow, DEAE Sepharose Fast Flow, SARTOBIND® Q, ANX Sepharose 4 Fast Flow (Hi Sub), Q Sepharose XL, Q Sepharose Big Bead, DEAE Sephadex A-25, DEAE Sephadex A-50, QAE Sephadex A-25, QAE Sephadex A-50, Q Sepharose High Performance, Q Sepharose XL, Source 15Q, Source 30Q, Resource Q, Capto Q, Capto DEAE, Mono Q, Toyopearl Super Q, Toyopearl DEAE, Toyopearl QAE, Toyopearl Q, Toyopearl Gigacap Q, TS Gel Super Q, TS Gel DEAE, Fracto Gel EMD TMAE, Fractogel EMD TMAE Hicap, Fractogel EMD DEAE, Fractogel EMD DMAE, Macroprep Hi-Q, Macro-Prep-DEAE, Unosphere Q, Nubia Q, PORGS PI, DEAE Ceramic Hyper-D, Includes Q Ceramic HyperD. Other examples include DEAE Cellulose, Poros® PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 (Applied Biosystems), Satobind® Q (Sartorius), MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX Sepharose® Fast Flow, Q Sepharose® High Performance, QAE SEPHADEX® and FAST Q Sepharose® (GE Healthcare), WP PEI, WP DEAM, WP QUAT (J. T. Baker), Hydrocell DEAE and Hydrocell QA (Biochrom Labs Inc.), Unosphere Q, Macro-Prep® DEAE and Macro-Prep® High Q (Biorad), Ceramic HyperD Q, Ceramic HyperD DEAE, Trisacryl® M and LS DEAE, Spherodex LS DEAE, QMA SPHEROSIL® LS, QMA Spherosil® M and MUSTANG® Q (Pall Technologies), Dowex® Fine Mesh Strong Base Type I and Type II Anion Resin and Dowex® Monosphere 77 Weak Base Anion (Dow Liquid Separations), INTERCEPT® Q Membrane, Matrex Cellufine® A200, A500, Q500 and Q800 (Millipore), Fractogel ® EMD TMAE, Fractogel® EMD DEAE and Fractogel® EMD DMAE (EMD), Amberlite® weak and strong anion exchangers types I and II, DOWEX® weak and strong anion exchangers types I and II, die Aion® Weak and Strong Anion Exchanger Types I and II, DUOLITE® (Sigma-Aldrich), TSK Gel Q and DEAE 5PW and 5PW-HR, Toyopearl® Super Q-650S, 650M and 650C, QAE- 550C and 650S, DEAE-650M and 650C (Tosoh), QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D or Express-Ion Q (Whatman), Sartobind® Q (Sartorius, NY, USA) corporation), but is not limited thereto.

In one embodiment, the first anion exchange chromatography in the purification method of the present invention may use a weak anion exchanger.

In any one of the above embodiments, the weak anion exchange resin may have a weak anion exchange group. For example, an anion exchange resin filled with a resin having a primary, secondary or tertiary amine, specifically, a secondary or tertiary amine as an exchange group may be used. Positively charged forms of the above amines are also included.

In any one of the embodiments described above, the primary anion exchange chromatography is PEI (Polyethyleneimine), PAB (Para-aminobenzyl), AE (amino ethyl), DEAE (diethyl aminoethyl) or DMAE (dimethyl aminoethyl) column It may be, specifically, it may be a DEAE column. One example may be DEAE Sepharose Fast Flow (FF), but is not limited thereto.

In one embodiment, the secondary anion exchange chromatography in the purification method of the present invention may use a weak anion exchanger.

In any one of the above embodiments, the weak anion exchange resin may have a weak anion exchange group. For example, an anion exchange resin filled with a resin having a primary, secondary or tertiary amine, specifically, a secondary or tertiary amine as an exchange group may be used. Positively charged forms of the above amines are also included.

In any one of the embodiments described above, the secondary anion exchange chromatography is PEI (Polyethyleneimine), PAB (Para-aminobenzyl), AE (amino ethyl), DEAE (diethyl aminoethyl) or DMAE (dimethyl aminoethyl) column It may be, specifically, it may be a DEAE column. One example may be DEAE Sepharose Fast Flow (FF), but is not limited thereto.

In the purification method of the present invention, the "loading" step is to load the column by allowing the fluid (feed) containing the target protein to flow through the inlet, and the feed comes into contact with an adsorbent or resin to bind a specific substance. steps to make it happen. Certain substances and other molecules that are not bound flow through the outlet with the fluid.

In one embodiment, the purification method of the present invention may include washing the column before and after the loading step, eluting the target material, regenerating the adsorbent, and/or equilibrating the column.

In the present invention, "buffering agent" refers to a substance that, by virtue of its presence in a solution, increases the amount of acid or alkali that must be added to cause a unit change in pH. Buffered solutions resist changes in pH by the action of acid-base conjugate components. A buffered solution can maintain a constant concentration of hydrogen ions such that the pH of the solution is within a physiological range. Buffer components may include, but are not limited to, organic and inorganic salts, acids and bases.

For example, a phosphate buffer, a citrate buffer, or an acetate buffer may be used as the column buffer. Specifically, the column buffer may be a phosphate buffer, for example sodium phosphate, but is not limited thereto. For example, the column buffer may include a salt. For example, the column buffer may include, but is not limited to, salts such as NaCl, KCl, NaOAc, (NH ₄ ) ₂ SO ₄ , Na ₂ SO ₄ and CH ₃ COONa.

In any one of the above embodiments, the first anion exchange chromatography may include regenerating the column using a buffer.

In any one of the above embodiments, the buffer used in the column regeneration step of the first anion exchange chromatography may be a phosphate buffer containing a salt. In any one of the above embodiments, the buffer used in the column regeneration step of the first anion exchange chromatography may include sodium phosphate and sodium chloride. In any one of the foregoing embodiments, the concentration of the sodium phosphate may be about 100 mM to 300 mM. In any one of the foregoing embodiments, the concentration of sodium chloride may be about 400 mM to about 600 mM. However, it is not limited thereto.

In any one of the above embodiments, the first anion exchange chromatography may include equilibrating the column using a buffer.

In any one of the above embodiments, the buffer used in the column equilibration step of the first anion exchange chromatography may be a phosphate buffer. In any one of the above embodiments, the buffer used in the column equilibration step of the first anion exchange chromatography may include sodium phosphate and sodium chloride. In any one of the above embodiments, the concentration of the sodium phosphate may be about 1 to 100 mM, specifically about 2 to 50 mM, or about 5 to 35 mM, but is not limited thereto. In any one of the foregoing embodiments, the concentration of sodium chloride may be about 100 mM to 600 mM, for example about 100 mM to 500 mM, 100 mM to 450 mM, 100 mM to 400 mM, 150 mM to 500 mM, 150 mM to 450 mM, 150 mM to 400 mM, 200 mM to 500 mM, 200 mM to 450 mM, 200 mM to 400 mM, 250 mM to 500 mM, 250 mM to 450 mM, 250 mM to 400 mM, or 300 mM to 400 mM. For example, it may be about 350 mM. However, it is not limited thereto.

The regeneration and equilibration steps may be performed prior to the loading step.

In any one of the above embodiments, the first anion exchange chromatography may include loading a solution containing a target protein. The target protein of the solution may be included in the form of an inclusion body, but is not limited thereto. The flow rate of the loading step may be appropriately selected, and may be, for example, at least about 50 cm/hr, 60 cm/hr, 70 cm/hr or more, but is not limited thereto. The amount of the solution containing the target protein to be loaded may be appropriately adjusted. For example, the amount of sample loaded into a unit resin may be about 1 to 20 v/v, but is not limited thereto.

In any one of the above embodiments, the first anion exchange chromatography may include performing in a negative mode.

In the present invention, the term "negative chromatography" is also called "negative chromatography" or "flow-through chromatography", and refers to a chromatography method in which a target protein does not adhere to a column but impurities adhere to the column. By performing the negative mode chromatography, the target protein can be separated from impurities by allowing the target protein to exist in the flow-through. Conversely, "positive chromatography" refers to a chromatography method in which a target protein binds to a column and impurities do not bind. The target protein may be obtained by performing positive chromatography and then eluting the target protein bound to the column.

By including the step of performing the first anion exchange chromatography in a negative mode, the content of impurities can be lowered.

In any one of the above embodiments, the target protein of the present invention may be included in the permeate of the first anion exchange chromatography.

In any one of the above embodiments, the purification method of the present invention may further include loading and circulating the permeate of the first anion exchange chromatography into the first anion exchange column again. . The circulation may be performed until the impurity content is lowered to a desired level. For example, the circulation volume may be about 2 times or more, 3 times or more, 4 times or more, 5 times or more, for example, about 10 times the resin volume, but is not limited thereto.

In any one of the above embodiments, the purification method of the present invention may include filtering the solution obtained in the first anion exchange chromatography step. The filtering step may include passing the permeate through a filter having a pore size of about 0.01 μm to about 0.5 μm, for example about 0.1 μm to about 0.3 μm, specifically about 0.2 μm. However, it is not limited thereto.

In any one of the above embodiments, the solution obtained through the first chromatography may be loaded into the second anion exchange column.

In any one of the above embodiments, the secondary anion exchange chromatography may be performed in a positive mode.

In any one of the above embodiments, the buffer of the secondary anion exchange chromatography may contain a detergent. The buffer includes the buffer of the equilibration step, the washing step and/or the elution step.

Examples of detergents included in the secondary anion exchange chromatography buffer include Triton X-100, Tween 80, Tween 20, sodium dodecyl sulfate (SDS), sodium tetradecyl sulfate, sodium hexadecyl sulfate, sodium deoxycholate , polyoxyethylene sorbitan monooleate, Brij, NP40, CHAPS, CHAPSO, or octaglucoside, specifically selected from the group consisting of Triton X-100, Tween 80, and Tween 20 detergent can be used. For example, Triton X-100 may be used. The amount of the detergent may be about 0.01% to 1%, or 0.5% to 0.05%, for example about 0.1% (v / v).

In any one of the above embodiments, the secondary anion exchange chromatography may include regenerating the column using a buffer.

In any one of the above embodiments, the buffer used in the column regeneration step of the secondary anion exchange chromatography may be a phosphate buffer containing a salt. In any one of the above embodiments, the buffer used in the column regeneration step of the secondary anion exchange chromatography may include sodium phosphate and sodium chloride. In any one of the foregoing embodiments, the concentration of the sodium phosphate may be about 100 to 300 mM. In any one of the foregoing embodiments, the concentration of sodium chloride may be about 400 mM to about 600 mM. However, it is not limited thereto.

In any one of the above embodiments, the secondary anion exchange chromatography may include equilibrating the column using a buffer.

In any one of the above embodiments, the buffer used in the column equilibration step of the secondary anion exchange chromatography may be a phosphate buffer. In any one of the above embodiments, the buffer used in the column equilibration step of the secondary anion exchange chromatography may include sodium phosphate and sodium chloride. In any one of the above embodiments, the concentration of the sodium phosphate may be about 1 to 100 mM, specifically about 2 to 50 mM, or 5 to 35 mM, but is not limited thereto. In any one of the above embodiments, the concentration of sodium chloride may be about 500 mM or less, for example about 400 mM or less, about 300 mM or less, or about 250 mM or less, for example, about 50 mM to 400 mM, about 50 mM to 300 mM, or about 50 mM to 250 mM. For example, it may be about 150 mM. However, it is not limited thereto.

The regeneration and equilibration steps may be performed before loading the solution containing the target protein obtained in the first chromatography into the second chromatography column.

In any one of the above-described embodiments, in the purification method of the present invention, the secondary anion exchange chromatography includes first washing the resin using a phosphate buffer containing a detergent and a salt; and secondarily washing the resin using a phosphate buffer containing detergent and salt.

The washing step may be performed after loading the solution obtained through the first chromatography and discarding the passing fraction.

In any one of the embodiments described above, the buffer in the first and second washing steps may be a phosphate buffer, specifically sodium phosphate. In any one of the above embodiments, the concentration of the sodium phosphate may be about 1 to 100 mM, specifically about 2 to 50 mM and 5 to 35 mM, but is not limited thereto.

In any one of the above-described embodiments, the buffer solution of the first and second washing steps may include sodium chloride. In any one of the foregoing embodiments, the concentration of sodium chloride in the first washing step buffer may be about 100 mM to 200 mM.

In any one of the embodiments described above, the concentration of sodium chloride in the second washing step buffer may be less than about 250 mM, specifically about 10 mM or more and less than 250 mM, 25 mM or more and less than 250 mM, 50 mM or more and less than 250 mM, 75 mM or more and less than 250 mM less than 100 mM and less than 250 mM, or greater than about 150 mM and less than 250 mM.

The second chromatography of the present invention may include the step of recovering the eluate by introducing a buffer solution after the washing step.

The elution buffer may be a phosphate buffer, specifically sodium phosphate. The buffer solution may include sodium chloride. The concentration of the sodium phosphate may be about 1 to 100 mM, specifically about 2 to 50 mM, or 5 to 35 mM, but is not limited thereto. The concentration of sodium chloride may be about 30 mM to 500 mM. For example, it may be less than 500 mM.

In any one of the above-described embodiments, the purification method of the present invention may include filtering the recovery solution of secondary anion exchange chromatography. The filtration step may include passing the recovered solution through a filter having a pore size of about 0.01 μm to about 0.5 μm, for example about 0.1 μm to about 0.3 μm, specifically about 0.2 μm. However, it is not limited thereto.

In the purification method of the present invention, in the step of performing the above-described chromatography, conductivity, flow rate, or flow rate may be appropriately adjusted.

In one embodiment, the purification method of the present invention may further include a filtration step after the chromatography step.

The filtration may be performed by conventionally known processes such as microfiltration, ultrafiltration, diafiltration, microfiltration, and depth filtration.

In any one of the above embodiments, the purification method of the present invention may include ultrafiltration and diafiltration processes after the chromatography step.

"Ultrafiltration" of the present invention refers to a membrane-based separation process that separates molecules in solution on the basis of size. Ultrafiltration processes are known in the art and are commonly used in protein purification processes (see, e.g., Zeman et al., Microfiltration and Ultrafiltration: Principles and Applications, Marcel Dekker, Inc., pp. 299-301 (1996) ). Ultrafiltration allows solvent and small solute molecules to pass through while macromolecules are retained, and thus can be used to purify and concentrate micromolecules such as proteins.

"Diafiltration" of the present invention is a type of ultrafiltration in which an aqueous buffer is added to the retentate, and an ultrafiltration membrane is used to remove salts from solutions containing proteins, peptides, nucleic acids, and other biomolecules. Or a method of removing or replacing the solvent or lowering its concentration.

In any one of the embodiments described above, the buffer used in the ultrafiltration and diafiltration steps is a salt selected from TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, Cacodylate, MES; and salts selected from NaCl, KCl, NaOAc, (NH4)2SO4, Na2SO4 and CH3COONa; and Triton X-100, Tween 80, Tween 20, sodium dodecyl sulfate (SDS), sodium tetradecyl sulfate, sodium hexadecyl sulfate, sodium deoxycholate, polyoxyethylene sorbitan monooleate, Brij, NP40 , CHAPS, CHAPSO, and octaglucoside. In any one of the above embodiments, the buffer may include Tris and sodium chloride. In any one of the foregoing embodiments, the detergent included in the buffer may be CHAPS. CHAPS, also called 3-cholamidopropyl dimethylammonio 1-propanesulfonate, is a non-denaturing zwitterionic detergent.

In any one of the above embodiments, the Tris may be included at a concentration of about 10 mM to 100 mM, specifically about 20 mM to 80 mM, 30 mM to 70 mM, or 40 mM to 60 mM, and one example may be included at a concentration of about 50 mM. there is.

In any one of the above embodiments, the sodium chloride may be included at a concentration of about 100 to 1000 mM, specifically about 200 mM to 800 mM, 300 mM to 700 mM, or 400 mM to 600 mM, and one example may be included at a concentration of about 500 mM. there is.

In any one of the foregoing embodiments, the CHAPS is about 0.01% to 5%, specifically 0.01% to 3%, 0.01% to 2%, 0.01% to 1%, 0.01% to 1.5%, 0.05% to 3%, 0.05% to 2%, 0.05% to 1.5%, 0.05% to 1%, 0.1% to 3%, 0.1% to 2%, 0.1% to 1.5%, 0.1% to 1%, 0.5% to 3% %, 0.5% to 2%, 0.5% to 1.5%, or 0.5% to 1% (v / v) may be included at a concentration, for example, it may be included at a concentration of about 0.75%.

A solution containing a target protein applied to the chromatography of the present invention may be provided as described below.

The target protein of the present invention can be recovered from prokaryotic or eukaryotic cells. For example, it can be produced in animal cells, Escherichia coli, yeast, insect cells, plant cells, and living animals through genetic recombination. For example, prokaryotic cells and eukaryotic cells may be transformed or transfected with an expression vector containing a nucleic acid encoding the target protein of the present invention. Such transformation or transfection of expression vectors includes standard bacterial transformation, calcium phosphate co-precipitation, electroporation, or liposome mediated transfection, DEAE dextran mediated transfection, polycationic mediated transfection or viral mediated transfection. This may be achieved through any technique known in the art, but is not limited thereto. (See, e.g., Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press); Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY])).

In one embodiment, the target protein may be recovered from a strain expressing the protein. In any one of the above embodiments, the strain may be a microorganism of the genus Escherichia, for example, Escherichia coli ( E. coli ). The E. coli may be, for example, E.coli BL21, JM109, K-12, LE392, RR1, DH5α or W3110 strain, but is not limited thereto.

In any one of the above embodiments, a step of culturing a strain expressing the protein in a medium containing glycerol may be included prior to the purification step in order to produce the target protein provided in the present invention. The strain may include a polynucleotide encoding the target protein of the present invention, or a vector containing the polynucleotide.

In any one of the above embodiments, the polynucleotide encoding the target protein provided in the present invention is the nucleotide sequence of SEQ ID NO: 2 or at least 70%, 75%, 80%, 85%, 86%, 87 It may have %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 94%, 95%, 96%, 97%, 98% or 99% or more identity, but is limited thereto. It doesn't work.

The polynucleotide encoding the target protein provided in the present invention can be used within a range that does not change the amino acid sequence of the protein due to codon degeneracy or considering codons preferred in organisms in which the protein is to be expressed. Various modifications may be made to the coding region.

In addition, the polynucleotide of the present invention hybridizes with a probe that can be prepared from a known gene sequence, for example, a complementary sequence for all or part of the nucleotide sequence under stringent conditions to encode the target protein of the present invention. Any sequence may be included without limitation.

The "stringent condition" means a condition that allows specific hybridization between polynucleotides. These conditions are described in the literature (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology , John Wiley & Sons, Inc., New York).

A polynucleotide encoding a protein of interest provided in the present invention can be engineered in a variety of ways to enable expression of the protein of interest. Depending on the expression vector, it may be necessary to manipulate the polynucleotide prior to inserting it into the vector. Such manipulations may use methods known in the art.

As used herein, the term "expression" includes, but is not limited to, any step involved in the production of a polypeptide, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

The "vector" of the present invention is a DNA preparation containing the nucleotide sequence of a polynucleotide encoding the target protein operably linked to a suitable expression control region (or expression control sequence) so as to express the target protein in a suitable host. it means. The expression control region may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome and can integrate into the genome itself.

Vectors that can be used in the present invention are not particularly limited, and any vectors known in the art can be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, and pBluescriptII-based plasmid vectors , pGEM-based, pTZ-based, pCL-based, pET-based, etc. can be used. Specifically, pET28, pET29, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used. For example, a polynucleotide encoding a target protein of the present invention can be inserted into a chromosome through a vector for chromosomal insertion into a cell. Insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker for determining whether the chromosome is inserted may be further included. Selectable markers are used to select cells transformed with a vector, that is, to determine whether a target nucleic acid molecule has been inserted, and to give selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface polypeptide expression. markers may be used. In an environment treated with a selective agent, only cells expressing the selectable marker survive or exhibit other expression traits, so transformed cells can be selected.

In any one of the above embodiments, the method for producing the target protein may include culturing the strain in a medium containing glycerol.

In any one of the above-described embodiments, the method for producing the target protein may further include adding an expression inducer to the medium after culturing the strain in a medium containing glycerol.

In any one of the above embodiments, the method for recovering the target protein from the strain may include disrupting the strain. For example, the recovery method may include suspending the strain into a suspension and crushing the suspension. The strain may be a microorganism of the genus Escherichia, and may be, for example, Escherichia coli ( E. coli ). The E. coli may be, for example, E.coli BL21, JM109, K-12, LE392, RR1, DH5α or W3110. However, it is not limited thereto.

Cell lysis methods include the use of alkalis, surfactants, organic solvents or enzymes (such as lysozyme), sonication or French Press, and periodic high temperature, pressure, freezing or thawing cycles. It can be done by applying

In any one of the above embodiments, impurities may be removed by treatment with nuclease after the step of disrupting the strain. The nuclease may be an endonuclease, and examples thereof include Serratia marcescens endonuclease (Benzonasae), Omnicleave (Epicentre), or DNase I.

In any one of the above embodiments, the target protein may be recovered in the form of an inclusion body. An inclusion body refers to an aggregate state in which polypeptides are gathered at high density due to misfolding. It is protein solubilization and refolding that unravels the inclusion body form and returns to the normal protein form. A solubilization buffer may be used to restore the target protein recovered in the inclusion body form to a normal protein form.

A multimeric protein assembly including the target protein of the present invention may also be referred to as a "nano structure (sieve)". The multimeric protein assembly may include an immunogenic fragment of the SARS-CoV-2 Spike protein. As used herein, the term "immunogenicity" refers to the ability of a specific protein or a specific region thereof to elicit an immune response against a specific protein or a protein comprising an amino acid sequence having a high degree of identity with the specific protein. do. The multimeric protein assembly may have a structure including 12 pentamers of the target protein of the present invention. Thus, the multimeric protein assembly of the present invention contains 60 copies of Component B.

A multimeric protein assembly comprising the protein of interest of the present invention can be used as a component of an immunogenic composition.

A target protein can be obtained with high purity through the purification method of the present invention.

Figure 1 confirms the impurity removal rate by adjusting the loading ratio, linear velocity, and circulation time of the first chromatography process.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

Example 1. Preparation of host cells expressing Component B

BL21 competent cells distributed by Thermofisher were transformed with the vector into which the Component B protein expression gene was introduced.

The vector (Genscript) into which the Component B protein expression gene was introduced was transferred from the Institute for Protein Design at the University of Washington (IPD) to SK bioscience and used to manufacture E. coli cell lines for research. As a vector for gene introduction, pET29b+ was used.

The prepared plasmid (SEQ ID NO: 3) was transformed into E. coli strain BL21, an E. coli strain having kanamycin resistance was selected, and the selected E. coli strain BL21 was inoculated into a soybean-based medium containing yeast extract at 200 rpm at 37 ° C. Incubation was performed with shaking until the absorbance reached 1.8 or higher. When the culture was completed, sterilized glycerol was added to a final concentration of 25% to construct a cell bank for research.

Example 2. Host cell culture

Example 2-1. Seed Fermentation

After dissolving 1 vial of cell bank for E. coli manufacturing at room temperature, add kanamycin solution to a final concentration of 30 mg/mL in a 1 L flask containing 500 mL of soybean-based medium containing yeast extract, and add 0.15 mL of the manufacturing strain. A seed culture was prepared by inoculation. At this time, the seed culture conditions were cultured at 37 ° C. with shaking at 200 rpm for more than 8 hours so that the final absorbance exceeded 1.5.

Example 2-2. main culture

Add kanamycin to a final concentration of 30 mg/mL in a 75 L fermentor containing 50 L of soybean-based medium containing yeast extract, inoculate the entire amount of seed culture medium, and incubate at 37°C at 200 rpm until the absorbance reaches 25. (Growth phase). When the absorbance reached 25, the fermentor settings were changed to a culture temperature of 18 ° C and an agitation speed of 150 rpm, IPTG was added to a final concentration of 0.5 mM, followed by induction phase for 10 hours, and then the main culture was terminated.

Example 3. Cell Recovery, Freezing, Disruption and Inclusion Body Isolation

After the main culture was completed, the cells were recovered using a high-speed centrifuge. At this time, the centrifugation conditions were performed at a centrifugation speed of 15,000 g and a centrifugation time of 20 minutes, and the culture supernatant was discarded and the cells were recovered. The recovered cells were stored in a cryogenic freezer below -70°C.

Using 10 mL of buffer per 1 g of cells, the mixture was stirred for 1 to 2 hours until the cells were completely released. The cell suspension was disrupted twice using a high-pressure disruptor at a pressure of 17,000 psi to recover the cell disruption solution. The recovered cell homogenate was treated with benzonase to a final concentration of 5 units/mL and reacted at room temperature for 3 hours.

Benzonase-treated cell lysate was centrifuged at 15,000 g for 60 minutes using a high-speed centrifuge, and the supernatant was discarded and inclusion bodies were recovered. The recovered inclusion bodies were washed twice using a sodium phosphate buffer solution, dissolved using a sodium phosphate buffer solution containing 2 M urea, and collected by 0.2 μm filtration.

Example 4. Purification of Component B by chromatography

Example 4-1. First Chromatography

First chromatography was performed using a column filled with diethylaminoethyl (DEAE) resin to remove host-derived impurities such as endotoxin and nucleic acid.

Specifically, the DEAE resin-packed column (DEAE sepharose FF) was regenerated using 200 mM sodium phosphate and 500 mM sodium chloride buffer, and equilibrated using 20 mM sodium phosphate and 350 mM sodium chloride buffer. The inclusion body recovered in Example 3 was introduced into the chromatography column after equilibration was completed at a flow rate of 100 cm/hr. After the introduction, when UV280 increased by 0.005 AU or more, collection of the permeate was started, and collection was stopped when UV280 value decreased again to reach equilibrium. The recovered permeate was filtered through 0.2 μm.

Example 4-2. Secondary chromatography

In the second chromatography, endotoxins and host-derived proteins remaining after the first chromatography were additionally removed using a column filled with diethylaminoethyl (DEAE) resin.

A column (DEAE sepharose FF) packed with DEAE resin was regenerated using 200 mM sodium phosphate and 500 mM sodium chloride buffer, and equilibrated using 20 mM sodium phosphate and 150 mM sodium chloride buffer. The first chromatographic permeate was introduced into the equilibrated chromatography column at a flow rate of 100 cm/hr, and the fraction that passed without being adsorbed to the resin was discarded. Again, the resin was washed first with a buffer containing 20 mM sodium phosphate, 150 mM sodium chloride, and 0.1% Triton X-100, and washed secondly with a buffer containing 20 mM sodium phosphate and 200 mM sodium chloride. At this time, all washing liquid was discarded. After washing was completed, a buffer solution containing 20 mM sodium phosphate and 400 mM sodium chloride was added, and the eluate was recovered when the UV280 value reached 0.01 AU or more, and the recovery was terminated when the UV280 value decreased again to reach equilibrium. The recovered solution was filtered through 0.2 μm.

Example 4-3. UF/DF

The secondary chromatography recovered solution was concentrated (Ultrafiltration) to 5 kg using a tangential flow filtration system equipped with a 100 kDa MWCO (Molecular weight cut-off) membrane with an area of 0.7 m ² , and It was recovered by performing buffer exchange (Diafiltration) using 50 mM Tris buffer. At this time, the transmembrane pressure (TMP) was maintained at 1 bar or less. The recovered solution was filtered through 0.2 μm.

In order to improve the impurity content, (i) application of the first chromatography (negative mode) and setting the process factor conditions, (ii) composition of the washing buffer and detergent addition in the second chromatography, (iii) after the second chromatography The buffer composition was adjusted during ultrafiltration.

Example 5. 1st Chromatography Process Improvement

Example 5-1. Identification of impurity content according to whether or not the first chromatography was performed

First, the change in the impurity level depending on whether the first chromatography was performed in a negative mode was confirmed. Group 1 was indicated when negative mode chromatography was not performed, and Group 2 was indicated when negative mode chromatography was performed. The overall purification conditions are as follows.

Group Chromatography application Product quality Negative mode Positive mode Endotoxin content (EU/mg of protein) Host cell DNA content (ng/mg of protein) Productivity (mg/L of fluid) Group 1 X O 11,924 2.9 62 Group 2 O O 517 1.6 89

As a result of the experiment, the protein yield and the amount of impurities were as shown in Table 1, and it was confirmed that the content of endotoxin and nucleic acid, which are impurities, was lowered by the addition of the first chromatography process performed in negative mode. did

Example 5-2. Optimization of conditions for performing primary chromatography

Next, a design of test (DoE) was performed to confirm the impurity removal rate by adjusting the loading ratio, linear velocity, and circulation time (CV) of the sample loaded into the unit resin during the first chromatography (Fig. 1 ). As a result of the experiment, it was confirmed that the yield increased at 10CV, which is a condition of reloading the flow-through of about 10 times the volume of the resin.

Example 6. Secondary chromatography process improvement

Example 6-1. Confirmation of purification yield according to the presence or absence of detergent in the secondary chromatography buffer

The purification yield was confirmed according to the presence or absence of detergent in the equilibration, washing and elution buffers used in the second chromatography. Other compositions in the buffer are as described in Example 4-2.

Group Test condition Product quality Triton concentration in buffer (%) Endotoxin content (EU/mg of protein) Host cell DNA content (ng/mg of protein) Productivity (mg/L of fluid) Group 1 0 343 1.7 48 Group 2 0.1 662 11.7 212

The experimental results are as shown in Table 2 above.

Through this, it was confirmed that protein productivity increased when detergent was used in the secondary chromatography at 0.1% content compared to when detergent was not included.

Example 6-2. Salt content adjustment of the second chromatography wash buffer

In the composition of the secondary chromatography secondary washing buffer, the NaCl content was changed to minimize the content of impurities and the composition that minimized the yield reduction was confirmed. Other compositions in the buffer are as described in Example 4-2.

As a result of the experiment, protein yield and impurity content are shown in Table 3 below.

Group Test condition Product quality NaCl (mM) in washing buffer Endotoxin content (EU/mg of protein) Host cell DNA content (ng/mg of protein) Productivity (mg/L of fluid) Group 1 0 25,100 6,466 127 Group 2 200 11,924 2.9 62 Group 3 250 11,099 17.2 10

Through this, it was confirmed that the optimal content of NaCl in the second washing buffer of the second chromatography was about 200 mM.

Example 7. Improvement of the ultrafiltration process

Example 7-1. Confirmation of optimal composition of buffer for ultrafiltration process

By changing the composition of the buffer solution in the ultrafiltration process, we tried to identify the buffer showing the optimal yield.

As a result of using the four compositions of Table 4 below, it was confirmed that the composition of Group 4 (50mM Tris, 500mM NaCl, 0.75% CHAPS (pH 7.5)) had the best yield.

Group Diafiltration buffer composition Process Yield (%) Whether or not aggregation occurred during diafiltration One 50 mM Tris + 5% sucrose +
100 mM Arginine + 150 mM NaCl 100 O 2 50mM Tris +
100 mM Arginine + 150 mM NaCl 120 O 3 50 mM Tris + 5% sucrose +
500 mM Arginine + 150 mM NaCl 136 O 4 50 mM Tris + 500 mM NaCl + 0.75% CHAPS 137 X

Specifically, in the case of Groups 1 to 3, precipitates were formed over time during storage, but Group 4 was confirmed to be stably maintained without precipitates.

Example 7-2. Confirmation of protein yield according to the type of detergent included in the buffer in the ultrafiltration process

The yield was confirmed by changing the type of detergent to PS80 and Triton in the composition of Group 4 selected through the above experiment.

Group composition Protein content reduction rate (%) One 50 mM Tris, 500 mM NaCl 21.6 2 50 mM Tris, 500 mM NaCl, 0.75% CHAPS 1.88 3 50 mM Tris, 500 mM NaCl, 0.05% PS80 8.81 4 50 mM Tris, 500 mM NaCl, 0.1% Triton 22.1

As a result of the experiment, it was confirmed that the protein content reduction rate was the lowest when using Group 2, that is, CHAPS.

Through this, it was confirmed that 50 mM Tris, 500 mM NaCl, and 0.75% CHAPS were the optimal composition of the ultrafiltration process buffer.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

<110> SK bioscience Co., Ltd. <120> A method for purification of target protein <130> KPA211708-KR <150> KR 10-2021-0137835 <151> 2021-10-15 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> Component B <400> 1 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asn Gly Met Met Asn Val Gln Leu Asn Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Asn Tyr Asp Lys Ser Lys Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 2 <211> 474 <212> DNA <213> Artificial Sequence <220> <223> Component B <400> 2 atgaaccagc acagtcacaa ggacacgaa acggtaagaa tagcggtagt gcgtgcgcgt 60 tggcatgcgg aaattgtgga cgcctgtgg agtgcgtttg aagccgcgat gcgtgatatt 120 ggcggcgatc gttttgccgt ggatgtgttt gatgtgccgg gtgcgtacga aattccactg 180 catgcgcgta ccctggcgga aaccggccgt tatggcgcgg tgttaggcac cgcctttgtg 240 gtgaatggtg gcatttatcg tcacgaattt gtggcgagcg cggttattaa cggcatgatg 300 aatgtgcagc tgaacacggg cgtgccagtg ttaagtgccg tgctgacccc acacaactat 360 gataaaagca aagcccatac cctgctgttc ttagcgctgt ttgcggtgaa aggcatggaa 420 gcggcgcgt g cctgcgtgga gattttagcg gcccgtgaaa agattgcggc gtga 474 <210> 3 < 211> 5712 <212> DNA <213> Artificial Sequence <220> <223> plasmid <400> 3 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttc gctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaat g agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg aattaattct tagaaaaact catcgag cat caaatgaaac tgcaatttat 600 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900 cgttatcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380 cccttgtatt actgtttatg taagcagaca gtttattgt tcatgacca aatccc ttaa 1440 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1 680 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacga cctac 1860 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100 gccttttta c ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520 gttttcaccg tcatcaccga aacgc gcgag gcagctgcgg taaagctcat cagcgtggtc 2580 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 270 0 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940 tgcgatgcag atccggaaca taatggtgca gggcg ctgac ttccgcgttt ccagacttta 3000 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagt catgc cccgcgccca 3420 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540 cctgtcgtgc cagctgcatt aatgaatcgg c caacgcgcg gggagaggcg gtttgcgtat 3600 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggta atg gcgcgcattg 3840 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260 tgt gcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380 gggccagact ggaggtggca acgccaatca gcaacgactg tttg cccgcc agttgttgtg 4440 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680 t ctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860 cgagcccgat cttcccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc 4980 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa 5040 ataattttgt ttaactttaa gaaggagata tacatatgaa ccagcacagt cacaaggacc 5100 acgaaacggt aagaatagcg gtagt gcgtg cgcgttggca tgcggaaatt gtggacgcct 5160 gtgtgagtgc gtttgaagcc gcgatgcgtg atattggcgg cgatcgtttt gccgtggatg 5220 tgtttgatgt gccgggtgcg tacgaaattc cactgcatgc gcgtaccctg gcggaaaccg 528 0 gccgttatgg cgcggtgtta ggcaccgcct ttgtggtgaa tggtggcatt tatcgtcacg 5340 aatttgtggc gagcgcggtt attaacggca tgatgaatgt gcagctgaac acgggcgtgc 5400 cagtgttaag tgccgtgctg accccacaca actatgataa aagcaaagcc cataccctgc 5460 tgttcttagc gctgtttgcg gtgaaaggca tggaagcggc gcgtgcctgc gtggagattt 5520 tagcggcccg tgaaaagatt gc ggcgtgac tcgagcacca ccaccaccac cactgagatc 5580 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac 5640 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa 5700 ctatatccgg at 5712 <210> 4 <211> 157 < 212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 4 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Ala Glu His His Arg 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 5 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 5 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Ala Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 6 <211> 157 <212> PRT <213> Artificial Sequence <220 > <223> synthetically constructed nanostructure polypeptide <400> 6 Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Gln Cys Val Arg Ala 20 25 30 Phe Glu Glu Ala Met Ala Asp Ala Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Ser Ser Arg Glu His Glu 115 120 125 Phe Phe Arg Glu His Phe Met Val Lys Gly Val Glu Ala Ala Ala Ala 130 135 140 Cys Ile Thr Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 7 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 7 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Glu His Arg 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 8 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400 > 8 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Glu Asp His Glu 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 9 <211> 157 < 212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 9 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 10 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> synthetically constructed nanostructure polypeptide <400> 10 Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala 20 25 30 Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Ala Asp Thr Leu 115 120 125 Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala 130 135 140 Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala 145 150 155 <210> 11 <211> 157 <212> PRT <213> Artificial Sequence <220 > <223> synthetically constructed nanostructure polypeptide <220> <221> MISC_FEATURE <222> (9) <223> Xaa is Tyr or His <220> <221> MISC_FEATURE <222> (82) <223> Xaa is Asn or Asp <220> <221> MISC_FEATURE <222> (87) <223> Xaa is Arg or Asp <220> <221> MISC_FEATURE <222> (105) <223> Xaa is Ser or Asp <220> <221> MISC_FEATURE < 222> (119) <223> Xaa is Arg or Glu <220> <221> MISC_FEATURE <222> (121) <223> Xaa is Arg or Glu <220> <221> MISC_FEATURE <222> (124) <223> Xaa is Ala or Asp <220> <221> MISC_FEATURE <222> (126) <223> Xaa is His or Asp <220> <221> MISC_FEATURE <222> (128) <223> Xaa is Arg or Glu <220> <221> MISC_FEATURE <222> (150) <223> Xaa is Ala or Asn <400> 11 Met Asn Gln His Ser His Lys Asp Xaa Glu Thr Val Arg Ile Ala Val 1 5 10 15 Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala 20 25 30 Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp 35 40 45 Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr 50 55 60 Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val 65 70 75 80 Val Xaa Gly Gly Ile Tyr Xaa His Glu Phe Val Ala Ser Ala Val Ile 85 90 95 Asp Gly Met Met Asn Val Gln Leu Xaa Thr Gly Val Pro Val Leu Ser 100 105 110 Ala Val Leu Thr Pro His Xaa Tyr Xaa Asp Ser Xaa Glu Xaa His Xaa 115 120 125 Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala 130 135 140 Cys Ile Glu Ile Leu Xaa Ala Arg Glu Lys Ile Ala Ala 145 150 155

Claims (19)

Using a solution containing the target protein of SEQ ID NO: 1 that was expressed in E. coli to dissolve the inclusion bodies and recovered,
(i) first anion exchange chromatography, and
(ii) performing a second anion exchange chromatography;
The primary and secondary anion exchange chromatography includes loading an anion exchange resin column filled with a resin having a secondary or tertiary amine as an exchange group,
The first anion exchange chromatography is in negative mode,
The second anion exchange chromatography includes performing in a positive mode,
The second anion exchange chromatography includes first washing the resin using a buffer containing detergent and salt; and secondarily washing the resin using a buffer containing detergent and salt;
The salt of the buffer in the second washing step is contained at a concentration of 150 mM or more and less than 250 mM,
Detergents included in the secondary anion exchange chromatography buffer are Triton X-100, Tween 80, Tween 20, sodium dodecyl sulfate (SDS), sodium tetradecyl sulfate, sodium hexadecyl sulfate, sodium deoxycholate, polyoxy A method for purifying a target protein selected from ethylene sorbitan monooleate, Brij, NP40, CHAPS, CHAPSO, and octaglucoside.
The method of claim 1, wherein the first anion exchange chromatography comprises equilibrating the column with a buffer containing a salt at a concentration of 100 mM to 500 mM,
Secondary anion exchange chromatography comprises the step of introducing an elution buffer containing a salt of 30mM to 500mM after the washing step, a method for purifying a target protein.
The method of claim 1, wherein the anion exchange resin column is a PEI (Polyethyleneimine), PAB (Para-aminobenzyl), AE (amino ethyl), DEAE (diethyl aminoethyl), or DMAE (dimethyl aminoethyl) column.
The method of claim 1, wherein the buffer is a phosphate buffer and the salt is sodium chloride.
delete The method of claim 1, wherein the detergent included in the secondary anion exchange chromatography buffer is selected from the group consisting of Triton X-100, Tween 80, and Tween 20.
The method of claim 1, wherein the amount of the detergent is 0.01 to 5% (v/v).
The method of claim 1, wherein the secondary anion exchange chromatography comprises first washing the resin using a phosphate buffer containing detergent and salt; and secondarily washing the resin using a phosphate buffer containing detergent and salt.
The method according to claim 8, wherein the salt contained in the buffer in the second washing step is contained at a concentration of 150 mM or more and less than 250 mM.
The method of claim 8, wherein the salt is sodium chloride (NaCl).
The method according to any one of claims 6 to 10, wherein the detergent is Triton X-100.
The method of claim 1, wherein the purification method further comprises filtering the solution recovered from the second chromatography.
The method of claim 12, wherein the filtration is ultrafiltration or diafiltration.
The method of claim 12, wherein the buffer of the filtration process (buffer) is Tris; sodium chloride; and Tween 80, Tween 20, sodium dodecyl sulfate (SDS), sodium tetradecyl sulfate, sodium hexadecyl sulfate, sodium deoxycholate, polyoxyethylene sorbitan monooleate, Brij, NP40, CHAPS, CHAPSO and A method for purifying a target protein comprising a detergent selected from octaglucoside.
The method of claim 14, wherein the detergent is CHAPS.
The method of claim 14 or 15, wherein the amount of the detergent is 0.01% to 5% (v/v).
The method of claim 1, wherein the purification method does not perform an additional chromatography process.
The method of claim 1, wherein the purification method uses a solution containing the target protein of SEQ ID NO: 1,
(i) performing primary anion exchange chromatography in negative mode using a column packed with a resin having a secondary or tertiary amine as an exchange group;
(ii) performing secondary anion exchange chromatography in positive mode using a column packed with a resin having a secondary or tertiary amine as an exchange group and an equilibration, washing and elution buffer containing Triton X-100; As, the washing is performed twice, the buffer of the second washing step contains a salt concentration of 150mM or more and less than 250mM; and
(iii) a method for purifying a protein of interest, comprising performing ultrafiltration and diafiltration in the presence of a buffer containing a detergent.
The method of claim 18, wherein the detergents (ii) and (iii) are included in an amount of 0.01 to 5% (v/v), and the detergent of (iii) is CHAPS.

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