Recombinant Protein Extraction Reagents

Abstract

To provide an extraction reagent that is capable of culturing a transformant obtained by transforming a coliform bacillus by using a vector including polynucleotide which encodes Protein A derived from the genus staphylococcus, and efficiently extracting the Protein A out of the cultured transformant, and a method for producing the Protein A by using the reagent.SOLUTION: An extraction reagent includes at least nonionic surfactant and urea or anionic surfactant.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a reagent capable of efficiently extracting Protein A derived from Staphylococcus genus from Escherichia coli capable of expressing said Protein A, and a method for producing said Protein A using said reagent. [Background technology]

[0002] As a method for culturing a transformant obtained by transforming Escherichia coli with a vector containing a polynucleotide encoding a protein and extracting the protein from the cultured transformant, a physical disruption method such as ultrasonic disruption treatment or French press treatment, and a chemical treatment method using a commercially available extraction reagent have been known. However, when attempting to industrially extract the protein using these methods, the physical disruption method has a problem that it is extremely expensive to install a disruption device such as an ultrasonic disruption device or a French press, and the chemical treatment method has a problem that a large amount of expensive extraction reagent is used.

[0003] In addition to the above-mentioned methods, methods using various surfactants are known as methods for extracting the protein, such as a method of extraction with a reagent containing a nonionic surfactant and an anionic surfactant (Patent Document 1), a method of extraction with a reagent containing a cationic surfactant (Patent Document 2), and a method of extraction with a reagent containing a nonionic surfactant and a cationic surfactant (Patent Document 3). However, when the protein is a highly hydrophobic protein such as Protein A derived from the genus Staphylococcus, there is a problem that efficient extraction cannot be performed. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2016-145172 A [Patent Document 2] JP 2006-320313 A [Patent Document 3] JP 2013-252099 A Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a reagent capable of efficiently extracting Protein A in a short time when transforming Escherichia coli with a vector containing a polynucleotide encoding Protein A derived from the genus Staphylococcus and culturing the transformant obtained, and extracting the Protein A from the cultured transformant, and to provide a method for producing the Protein A using the reagent. [Means for solving the problem]

[0006] As a result of intensive research to solve the above problems, the present inventors have found that Protein A derived from the genus Staphylococcus (hereinafter also referred to as SpA) can be efficiently extracted from transformants in a short period of time by adding at least a nonionic surfactant and urea or an anionic surfactant to an extraction reagent.

[0007] That is, the present invention is as follows: <1> from <6> This includes aspects described in: <1> A reagent for extracting a recombinant protein expressed in Escherichia coli capable of expressing the protein, comprising: the recombinant protein is Protein A derived from the genus Staphylococcus; The reagent comprises at least a nonionic surfactant and, as an additive, urea or an anionic surfactant. <2> The additive is urea with a final concentration of 0.5 mol / L to 3 mol / L. <1> The reagent according to claim 1. <3> The additive is sodium dodecyl sulfate at a final concentration of 0.3 (w / v)% to 2 (w / v)%. <1> The reagent according to claim 1. <4> Further comprising a carbohydrate degrading enzyme, <1> from <3> The reagent according to any one of the preceding claims. <5> Further comprising a nuclease, <1> from <4> The reagent according to any one of the preceding claims. <6> Culturing Escherichia coli capable of expressing Staphylococcus protein A; and extracting the Protein A from the cells of the cultured E. coli using an extraction reagent containing at least a nonionic surfactant and urea or an anionic surfactant. A method for producing Protein A.

[0008] The present invention will be described in detail below.

[0009] The extraction reagent of the present invention is a reagent capable of extracting a protein expressed in an Escherichia coli transformant obtained by transforming the transformant with a vector containing a polynucleotide encoding a protein, without mechanical disruption treatment, by dissolving the cell wall of the transformant, and is characterized by containing at least a nonionic surfactant and at least urea or an anionic surfactant.

[0010] The nonionic surfactant contained in the extraction reagent of the present invention can be appropriately selected from among those commonly used as membrane protein solubilizing agents, and examples thereof include Triton X-100 (trade name), Triton X-114 (trade name), Brij 58 (trade name), Brij 35 (trade name), Tween 20 (trade name), Tween 80 (trade name), 1-On-octyl-β-D-glucopyranoside, n-octyl-β-D-thioglucopyranoside, n-dodecyl-β-D-maltopyranoside, n-dodecyl-α-D-maltopyranoside, n-dodecyl-N,N-dimethylamine-N-oxide, isopropyl-β-D-thiogalactoside, sucrose monododecanoic acid, n-octyl-β-D-glucopyranoside, n-dodecyl-β-D-maltopyranoside, n-tridecyl-β-D-maltopyranoside, etc. The concentration of the nonionic surfactant contained in the extraction reagent of the present invention may be appropriately set in consideration of the critical micelle concentration of the surfactant used, the properties and concentrations of impurities in the culture solution such as cell fragments, etc. As an example, when Triton X-100 is used as the nonionic surfactant, the final concentration is preferably set in the range of 0.2 (w / v) % to 2 (w / v) %.

[0011] Urea may be used as an additive contained in the extraction reagent of the present invention. Urea is generally used as a denaturant in the field of protein engineering, and has the effect of destroying the higher-order structure of proteins. It is preferable to set the urea concentration to a final concentration in the range of 0.5 mol / L to 3 mol / L, since this does not inactivate the recombinant protein expressed in E. coli, and only destroys the membrane structure of E. coli. It is more preferable to set the urea concentration to a final concentration in the range of 0.7 mol / L to 2.5 mol / L, and even more preferable to set the urea concentration to a final concentration in the range of 1 mol / L to 2 mol / L.

[0012] When an anionic surfactant is used as an additive contained in the extraction reagent of the present invention, it may be appropriately selected from among those commonly used as membrane protein solubilizers. Examples include sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, sodium cholate, and sodium deoxycholate. The concentration of the anionic surfactant may be appropriately set, as with the nonionic surfactant, taking into consideration the critical micelle concentration of the surfactant used, the properties and concentrations of impurities in the culture solution such as cell lysates, etc. As an example, when SDS is used as the anionic surfactant, the final concentration is preferably set in the range of 0.3 (w / v)% to 2 (w / v), and more preferably set in the range of 0.5 (w / v)% to 1.5 (w / v).

[0013] It is preferable to further include a carbohydrate degrading enzyme in the extraction reagent of the present invention, since this promotes the lysis of the transformant (Escherichia coli). The carbohydrate degrading enzyme that may be included in the extraction reagent of the present invention can be appropriately selected from among those commonly used as membrane protein solubilizing agents, and examples thereof include lysozyme, cellulase, pectinase, and salts thereof. The concentration of the carbohydrate degrading enzyme may be appropriately set in consideration of the properties and concentrations of impurities in the culture solution, such as cell lysates. As an example, when human-derived lysozyme is used as the carbohydrate degrading enzyme, the final concentration may be set in the range of 0.0001% (w / v) to 0.01% (w / v).

[0014] The extraction reagent of the present invention may contain components other than the above-mentioned components. In particular, it is preferable to further contain an endonuclease (nuclease) such as Benzonase (Merck), since this can suppress the increase in viscosity caused by nucleic acids extracted from the transformant together with proteins by the above-mentioned extraction reagent. When the extraction reagent of the present invention further contains a nuclease, it is preferable to contain magnesium sulfate at a final concentration of about 2 mmol / L as an auxiliary agent.

[0015] In addition to the nuclease, other components that may be contained in the extraction reagent of the present invention include buffer components and salts. Buffer components include acetate buffers with concentrations of about 10 mmol / L to 100 mmol / L, citrate-phosphate buffers, phosphate buffers, Tris (Tris(hydroxymethyl)aminomethane)-HCl buffers, glycine-NaOH buffers, MES (2-Morpholinoethanesulfonic acid) buffers, ADA (N-(2-Acetamido)iminodiacetic acid) buffers, PIPES (Piperazine-1,4-bis(2-ethanesulfonic acid)) buffers, ACES (N-(2-Acetamido)-2-aminoethanesulfonic acid) buffers, Collamine-HCl buffers, BES (N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid) buffers, TES (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) buffers, HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic Examples of the buffer include glycine (N-[Tris(hydroxymethyl)methyl]glycine] buffer, glycineamide buffer, and bicine (N,N-Bis(2-hydroxyethyl)glycine) buffer. Examples of the preferred buffer include phosphate buffer or Tris-HCl buffer with a concentration of 20 mmol / L to 50 mmol / L and a pH of 6.0 to 8.0. Examples of the salt include sulfate, phosphate, acetate, citrate, carbonate, chloride, etc. with a final concentration of 10 mmol / L to 1000 mmol / L.

[0016] When extracting a protein from a transformant obtained by transforming Escherichia coli with a vector containing a polynucleotide encoding a recombinant protein using the extraction reagent of the present invention, it is preferable to perform the extraction operation under pH conditions (specifically, pH 5.0 to 9.0) at which the protein is not inactivated and the additionally added enzymes (carbohydrate degrading enzymes and nucleases) are active, since this improves the efficiency of protein extraction. In particular, it is more preferable to set the pH range to 5.5 to 6.5.

[0017] After extracting the protein from the transformant using the extraction reagent of the present invention, a purification procedure is performed to remove the extraction residue (cell debris, etc.) using a method known in the art, thereby obtaining the protein with a high purity. An example of the purification procedure is separation and purification using liquid chromatography. Examples of liquid chromatography include ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and affinity chromatography. By performing the purification procedure using a combination of these chromatographies, the protein with a high purity can be produced.

[0018] In addition, when performing purification operation using liquid chromatography, if the turbidity of the protein solution obtained in the extraction step is high (i.e., the clarity is low), problems such as deterioration of the protein separation ability in chromatography and reduction in reproducibility of column performance occur, so the higher the clarity of the protein solution obtained in the extraction step, the more preferable it is. Therefore, it is recommended to increase the clarity of the protein solution in advance by centrifugation or filtration before performing purification operation using chromatography. Methods for increasing the clarity of the protein solution by centrifugation include batch-type centrifugation using a dedicated bottle, and continuous centrifugation such as cylindrical type and separation plate type. Methods for increasing the clarity of the protein solution by filtration include a method using an MF membrane (microfiltration membrane) or UF membrane (ultrafiltration membrane), and a method using pressure filtration in combination with a filter aid. There are no particular limitations on the type of membrane used for filtration (material, pore size, molecular weight cutoff, etc.) as long as it can efficiently remove insoluble matter.

[0019] The method for analyzing the protein obtained by the production method of the present invention is not particularly limited as long as it can stably and efficiently quantify the protein from the culture medium, extract, etc., and examples of the method include ELISA (enzyme-linked immunosorbent assay) and Western blot.

[0020] SpA extracted using the extraction reagent of the present invention is not particularly limited as long as it can be expressed in the cells of a transformant obtained by transforming Escherichia coli with a vector containing a polynucleotide encoding the SpA. Examples include the full length of Protein A derived from Staphylococcus aureus, the immunoglobulin binding domains (domain C, domain E, domain D, domain A, domain B / Z) constituting the Protein A, and a protein having an amino acid substitution at a specific position in the amino acid sequence of the full length or the domain (hereinafter, also referred to as modified SpA). An example of Domain C of Protein A derived from Staphylococcus aureus is the amino acid residues from 270 to 327 of GenBank No. AAA26676 (SEQ ID NO: 1).

[0021] The SpA may contain only one or more of the full-length, domain, or modified SpA. The SpA may contain, for example, two or more, three or more, four or more, or five or more of the full-length, domain, or modified SpA, or ten or less, seven or less, five or less, four or less, three or less, or two or less, or any compatible combination thereof. When the SpA contains a plurality of domains and / or modified SpAs, the amino acid sequences of the domains and / or modified SpAs may or may not be identical. The domains and / or modified SpAs may be linked to each other, for example, via a suitable linker.

[0022] A preferred embodiment of the SpA is a polypeptide comprising one or more amino acid sequences set forth in SEQ ID NO: 2. The polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, provided that the amino acid sequence has the following amino acid substitutions (1) to (14): (1) The third asparagine in SEQ ID NO:1 is replaced with histidine (2) The fourth lysine in SEQ ID NO:1 is replaced with arginine (3) Substitution of valine for lysine at position 7 of SEQ ID NO:1 (4) Substitution of asparagine at position 11 of SEQ ID NO:1 with arginine (5) Substitution of glutamic acid at position 15 of SEQ ID NO:1 with alanine (6) Substitution of asparagine at position 21 of SEQ ID NO:1 with tyrosine (7) Substitution of glycine at position 29 of SEQ ID NO: 1 with alanine (8) Substitution of lysine at position 35 of SEQ ID NO:1 with arginine (9) Substitution of serine at position 39 of SEQ ID NO: 1 with glycine (10) Substitution of valine at position 40 of SEQ ID NO:1 with alanine (11) Substitution of lysine at position 42 of SEQ ID NO:1 with leucine (12) Substitution of lysine at position 49 of SEQ ID NO:1 with methionine (13) The 50th lysine in SEQ ID NO:1 is replaced with tryptophan (14) The lysine at position 58 of SEQ ID NO: 1 is replaced with aspartic acid. Effect of the Invention

[0023] The extraction reagent of the present invention is characterized by containing at least a nonionic surfactant and urea or an anionic surfactant. The extraction reagent of the present invention enables efficient extraction in a short time of Protein A expressed in a transformant obtained by transforming Escherichia coli with a vector containing a polynucleotide encoding Protein A derived from the genus Staphylococcus.

[0024] The extraction reagent of the present invention is particularly effective in extracting highly hydrophobic proteins such as Protein A derived from the genus Staphylococcus.

[0025] Furthermore, the method for extracting Protein A using the extraction reagent of the present invention is capable of extracting Protein A inexpensively and efficiently compared to methods that involve physical disruption or methods that use commercially available extraction reagents, and therefore can be said to be useful in the industrial production of Protein A derived from the genus Staphylococcus. [Brief description of the drawings]

[0026] [Figure 1] FIG. 13 is a diagram showing the results of examining the urea concentration contained in an extraction reagent. EXAMPLES

[0027] The present invention will be described in more detail below using examples and comparative examples, but the present invention is not limited to these examples.

[0028] Example 1: Examination of additives (1) E. coli W3110 strain was transformed with an expression vector containing a polynucleotide (SEQ ID NO: 4) encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 to obtain a recombinant E. coli. The recombinant E. coli was cultured with reference to the method described in JP 2013-085531 A, and E. coli cells (wet cells) were obtained from the obtained culture solution by centrifugation. The polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 is an embodiment in which six Staphylococcus genus-derived Protein A (hereinafter also referred to as SpA) consisting of the amino acid sequence set forth in SEQ ID NO: 2 are directly linked together.

[0029] (2) The wet cells obtained in (1) were suspended in 20 mmol / L phosphate buffer (pH 6.0) containing 1 mmol / L EDTA in an amount three times the weight of the wet cells, and stirred at room temperature (20°C to 25°C) until homogenous.

[0030] (3) The bacterial cell suspension obtained in (2) was dispensed into a 96-well microwell plate in 500 μL portions, and then Benzonase (Merck), a nucleic acid degrading enzyme, was added to each well to a final concentration of 250 unit / L. Magnesium sulfate was added as an auxiliary agent to a final concentration of 2 mmol / L, and the mixture was stirred at room temperature.

[0031] (4) After adding any of the additives (a) to (n) below (all concentrations are final concentrations), lysozyme, a carbohydrate-degrading enzyme, was added to a final concentration of 0.005 (w / v)%, and the mixture was further stirred at room temperature.

[0032] (a) β-mercaptoethanol 1(v / v)% (b) β-mercaptoethanol 5(v / v)% (c) Urea 1mol / L (d) Urea 2mol / L (e)Tween 20 (product name) 0.1(v / v)% (f)Tween 20 (product name) 0.25(v / v)% (g)Tween 20 (product name) 1(v / v)% (h) Sodium dodecyl sulfate 0.1 (w / v)% (i) Sodium dodecyl sulfate 1(w / v)% (j) Sodium chloride 0.3 mol / L (k) Sodium chloride 1 mol / L (l) Sodium sulfate 0.1 mol / L (m) Arginine hydrochloride 1 mol / L (n) Guanidine hydrochloride 1 mol / L (5) An aqueous solution of Triton X-100 (product name), a nonionic surfactant, was added to a final concentration of 0.5 (w / v)%, and the mixture was stirred at room temperature to extract SpA.

[0033] (6) After performing the extraction procedure overnight, the extract from (5) was centrifuged (3000 rpm, 20 minutes, twice) to obtain the supernatant (cell-free extract).

[0034] (7) The amount of SpA extracted from the cell-free extract obtained in (6) was measured by the ELISA method described below. (7-1) A gamma globulin preparation, which is a human antibody (manufactured by The Chemical and Serum Therapeutic Research Institute), was immobilized in the wells of a 96-well microplate at a concentration of 10 μg / well (at 4° C. for 18 hours or more). (7-2) After immobilization, the plate was washed with a washing buffer (20 mmol / L Tris-HCl buffer (pH 7.5) containing 0.05 (w / v)% Tween 20 (trade name) and 150 mmol / L NaCl) and then blocked with 2 (w / v)% skim milk solution (Becton Dickinson and Company) (overnight at 4°C). (7-3) After washing with the washing buffer, the prepared cell-free extract was appropriately diluted with 50 mmol / L Tris-HCl buffer (pH 7.4) and reacted with immobilized gamma globulin (at 30° C. for 1 hour). (7-4) After the reaction was completed, the plate was washed again with the washing buffer, and Horse radish peroxidase (HRP)-labeled Anti-Protein A antibody reagent (Abcam) was added. (7-5) After reacting at 30°C for 1 hour, the mixture was washed with a washing buffer, TMB Peroxidase Substrate (KPL) was added, and after reacting for a desired period of time, a 1 mol / L aqueous phosphoric acid solution was added to stop the reaction, and the absorbance at 450 nm was measured.

[0035] Comparative Example 1 The same procedure as in Example 2 was carried out, except that no additive was added in Example 1(4).

[0036] Comparative Example 2 (1) SpA was extracted from the cell suspension obtained in Example 1(2) using a commercially available extraction reagent (BugBuster, manufactured by Merck) according to the standard protocol attached to the reagent.

[0037] (2) The extract was centrifuged to obtain the supernatant (cell-free extract).

[0038] (3) The amount of SpA extracted was measured by the method described in Example 1(7).

[0039] The results are shown in Table 1. The extraction amount was evaluated as "good" when the amount extracted was greater than that of the commercially available extraction reagent (Comparative Example 2), as "△" when the amount extracted was less than that of the commercially available extraction reagent (Comparative Example 2) but greater than that of the reagent without additive (Comparative Example 1), and as "poor" when the amount extracted was less than that of the reagent without additive (Comparative Example 1). The separation ability was evaluated as "good" when the interface between the supernatant and the precipitate was clearly separated by centrifugation, and only the supernatant was easily recovered without any mixture of the precipitate, and as "poor" otherwise.

[0040] [Table 1]

[0041] Table 1 shows that when urea was added at a final concentration of 1 mol / L (Table 1(c)) or 2 mol / L (Table 1(d)), and the anionic surfactant sodium dodecyl sulfate (SDS) was added at a final concentration of 1 (w / v)% (Table 1(i)), the amount of extraction was superior to that of commercially available extraction reagents, and the separation of the extract was also high. This shows that SpA expressed in E. coli can be extracted efficiently in a short time using an extraction reagent in which urea or an anionic surfactant is added as an additive to a nonionic surfactant.

[0042] Example 2: Urea concentration study (1) The same procedures as in Example 1(1) and (2) were carried out to obtain a cell suspension.

[0043] (2) To the bacterial cell suspension obtained in (1), Benzonase (Merck), a nuclease, was added to a final concentration of 250 units / L, and magnesium sulfate was added as an auxiliary agent to a final concentration of 2 mmol / L, followed by stirring at room temperature (20°C to 25°C).

[0044] (3) The bacterial suspension obtained in (2) was dispensed into five beakers at 20 mL each, and then an aqueous urea solution was added to each beaker to give a final concentration of 0.1, 0.5, 1.0, 2.0, or 3.0 mol / L. Lysozyme was then added to give a final concentration of 0.005 (w / v)%, and the mixture was stirred at room temperature (20°C to 25°C).

[0045] (4) An aqueous solution of Triton X-100 (product name), a nonionic surfactant, was added to a final concentration of 0.5 (w / v)%, and the mixture was stirred at room temperature (20°C to 25°C) to extract SpA.

[0046] (5) After the extraction procedure was carried out for 3 hours, the extract was centrifuged (9000 rpm, 20 minutes) to obtain the supernatant (cell-free extract).

[0047] (6) The amount of SpA extracted was measured by the method described in Example 1(7).

[0048] The results are shown in Figure 1. The addition of urea at a final concentration of 0.5 mol / L to 3 mol / L improved the amount of SpA extracted compared to when urea was not added. In particular, when urea at final concentrations of 1 mol / L and 2 mol / L was added, the amount of extraction was significantly improved compared to the commercially available extraction reagent (Comparative Example 2). This shows that SpA expressed in E. coli can be extracted efficiently in a short time by adding urea at a final concentration of 0.5 mol / L to 3 mol / L, preferably 0.7 mol / L to 3 mol / L, more preferably 1 mol / L to 2 mol / L. [Industrial Applicability]

[0049] According to the present invention, recombinant Protein A derived from the genus Staphylococcus expressed in Escherichia coli can be efficiently extracted in a short time. Therefore, the Protein A can be efficiently produced, which includes a step of culturing a transformant obtained by transforming Escherichia coli with a vector containing a polynucleotide encoding the Protein A, and a step of extracting the Protein A from the cultured transformant.

Claims

1. A reagent for extracting a recombinant protein expressed in Escherichia coli capable of expressing the protein, comprising: the recombinant protein is Protein A derived from the genus Staphylococcus; containing Triton X-100 (trade name) at a final concentration of 0.5 (w / v)%, The above reagent further comprises at least urea at a final concentration of 1 mol / L to 2 mol / L or sodium dodecyl sulfate at a final concentration of 1 (w / v)%.

2. The reagent of claim 1 , further comprising a glycolytic enzyme.

3. The reagent according to claim 1 or 2, further comprising a nuclease.

4. Culturing Escherichia coli capable of expressing Staphylococcus protein A; and extracting the Protein A from the cultured E. coli cells using an extraction reagent containing Triton X-100 (trade name) at a final concentration of 0.5 (w / v)% and further containing at least urea at a final concentration of 1 mol / L to 2 mol / L or sodium dodecyl sulfate at a final concentration of 1 (w / v)%. A method for producing Protein A.

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