JP2012050387A - Method for immobilizing two or more kinds of molecule in porous carrier - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide in large quantity and in a stable quality a column packing agent where a protein and molecules having functions different from those of the protein are immobilized in an activated porous carrier.SOLUTION: The column packing agent wherein the protein and molecules having functions different from those of the protein are immobilized in a porous carrier are prepared with stable quality, even in case of preparing a large quantity of the packing agent, when the protein and the molecules having functions different from those of the protein are immobilized in the porous carrier, by conducting a continuous one-pot reaction even when the replacing operation of the reaction liquid is skipped during the immobilizing reaction.

Description

  The present invention relates to a method for synthesizing a column filler, wherein a protein and a molecule having a function different from that of the protein are immobilized on a porous carrier in a continuous one-pot reaction.

  Immobilized proteins have been used in which proteins that can be used in various applications such as analysis and catalysis are immobilized on a carrier. However, in order to immobilize the carrier while maintaining the function of the protein, it is necessary to perform a special activation treatment on the carrier in advance. However, the time during which the carrier activation lasts during the immobilization reaction is short, and if two or more types of proteins are to be immobilized, the ability to immobilize the carrier is reduced. Often, desirable results are not obtained. In addition, an enzyme-immobilized reactor (bioreactor) in which an enzyme such as a protease used for various applications is immobilized on a carrier for use in the reaction has been proposed. However, with regard to protease, the immobilized protease is liable to be inactivated by decomposition due to autolysis. In addition, when the substrate of an enzyme such as a protease is a polymer, the reaction between the enzyme bound to the carrier and the substrate does not proceed efficiently and quickly due to spatial limitations. For these reasons, the use of enzyme-immobilized reactors (bioreactors) is limited depending on the type of enzyme and substrate used, and development of enzyme-immobilized reactors (bioreactors) that can be widely used for various enzymes and substrates. Is expected. In addition, as one of the enzyme-immobilized reactors (bioreactors), an affinity trap reactor has been developed that combines the separation method using affinity using an affinity column and the characteristics of enzyme reaction using a bioreactor column. Development of an affinity trap reactor that produces a substance that inhibits angiogenesis by using a derived protein as a substrate is also underway.

  Substances that inhibit angiogenesis are expected to be used as antitumor agents because they suppress cancer growth, invasion, and metastasis. Angiostatin is known as one of such substances. Yes. Angiostatin is a protein with a molecular weight of about 40,000 obtained by degrading plasminogen, a fibrinolytic factor, present in blood and has a dramatic effect on cancer in animal experiments. Has been reported (see Non-Patent Document 1). For the production of angiostatin, (1) plasminogen is hydrolyzed with a protease called elastase (see Non-patent Document 2), (2) it is directly produced in E. coli using a gene recombination technique (see Non-Patent Document 3). ), Two types of methods are used. In the method (1), since the substrate specificity of elastase is low, many by-products are produced, and it is difficult to selectively produce angiostatin from plasminogen. There are also disadvantages such as low activity of the resulting angiostatin. In the method (2), purification of angiostatin produced by Escherichia coli is difficult and costly. There is also a problem with low solubility. For this reason, development of a means for obtaining highly pure angiostatin by a simple method has been demanded.

  Recently, Bacilolysin MA, which is a protease produced by Bacillus megaterium A9542 strain, has been produced by angiostatin-like fragment that specifically cleaves plasminogen and has an antiangiogenic action (mainly Glu1-Ser441, hereinafter BL-angiostatin and Described) and was found to produce a miniplasminogen-like fragment (primarily Val442-Asn791) with thrombolytic activity (see Patent Document 1). Moreover, since this enzyme basilolysin MA is very stable, it can be immobilized on a carrier and used for various applications. Therefore, using such characteristics of basilolysin MA, the steps from the reaction of basilolysin MA to the substrate plasminogen and the purification of the resulting product BL-angiostatin are carried out in a single step with high purity. There was a need to develop a method for obtaining angiostatin and a device for doing so.

  In order to solve the above-mentioned problems, basilolysin MA is immobilized as a protease (enzyme) on a commercially available Sepharose-based activation carrier, and lysine (Lys) is used as a molecule that specifically binds to the enzyme substrate. By using an affinity trap reactor, which is an immobilized column, a method for decomposing and purifying BL-angiostatin from plasminogen contained in a biological sample such as blood with high efficiency and speed has been developed (Patent Documents 2 and 3). reference).

  However, when producing a small amount of an affinity trap reactor using an activated carrier as described above, this is not a problem. However, if an attempt is made to produce a large amount of an affinity trap reactor at a time, BL-angio from plasminogen is produced. It has been found that there is a concern about the reproducibility of the performance of the affinity trap reactor due to the phenomenon that the purification efficiency of statin is greatly reduced.

  In addition, the same can be considered in the production of a bioreactor that immobilizes two or more types of enzymes using an activated carrier. If reproducibility of enzyme immobilization in a bioreactor is considered to be an important factor, Decreasing the reproducibility of quality due to scale-up becomes a major problem, and therefore it has been desired to develop a synthetic formulation with stable quality.

  The present inventor has conducted intensive research to set up synthesis conditions that stabilize the purification efficiency of BL-angiostatin even when producing a large amount of the affinity trap reactor as described above, and as a result, it is effective in stabilizing the quality. The present inventors have completed the present invention by discovering the preparation conditions of a certain affinity trap reactor.

JP 2002-272453 A Japanese Patent No. 3830501 WO2005 / 0779835

M.S.O'Reilly et al., “Cell” (USA), October 21, 1994, Vol. 79, No. 2, p315-328 M.S.O'Reilly et al., “Nature Medicine” (USA), 1996, Vol. 2, p. 689-692 B.K.Sim et al., “Cancer Research” (USA), 1997, Vol. 57, pp. 1329-1334

  When immobilizing basilolysin MA and lysine on an NHS-activated porous carrier, the present inventors greatly reduce the purification efficiency of BL-angiostatin when trying to obtain a large amount of column packing material. It was confirmed that the phenomenon was seen. That is, an object of the present invention is to provide a large amount and a stable quality of a column packing material in which a protein and a molecule having a function different from that of the protein are immobilized on a porous carrier.

  As a result of intensive studies to solve the above problems, the present inventors have found that the stability or decomposition rate of the active group during the immobilization reaction affects the stabilization of the quality of the column packing material. I understood. Therefore, even if a large amount of filler is prepared by omitting the replacement of the reaction solution and suppressing the deactivation of the active group, the protein and molecules having different functions from the protein are porous. It was clarified that a column packing immobilized on a carrier can be prepared. That is, the present invention relates to a method for synthesizing a column filler, wherein a protein and a molecule having a function different from that of the protein are immobilized on a porous carrier in a continuous one-pot reaction. Unless otherwise specified, the one-pot reaction means that the reaction of two or more steps is continuously performed without performing an operation such as replacement of the reaction solution.

  By immobilizing a protein and a molecule having a function different from that of the protein in a continuous one-pot reaction, a large amount of filler is provided with a stable quality.

Immobilization method of the present invention The immobilization method of the present invention is a method of immobilizing a protein and a molecule having a function different from that of the protein by a continuous one-pot reaction, and is performed in two conventional steps. It is possible to provide an immobilization technique for avoiding a decrease in reproducibility at the time of scale-up in the conventional immobilization method.

  The protein that can be immobilized by the above-described immobilization method and the molecule having a function different from that of the protein include a protein that can be immobilized on a porous carrier by a covalent bond and a molecule having a function different from that of the protein. For example, various proteins and molecules having functions different from the proteins can be used as necessary.

  For example, when an enzyme is immobilized as a protein, a hydrolase, a oxidoreductase, a transferase, a removal addition enzyme, an isomerase, a synthase, etc., including a protease, a saccharide-degrading enzyme, and a lipolytic enzyme, Various enzymes can be used. As a molecule having a function different from that of the protein, a protein, a saccharide, a polymer, a small molecule and the like can be selected. However, when the protein to be immobilized and the combination of molecules having a function different from that of the protein have a relationship such as an enzyme and a substrate, the function as a bioreactor can be sufficiently obtained by digesting either of them. Since it is not, it is not preferable.

  In addition, by combining a protein other than an enzyme as a protein and a saccharide or a low molecule as a molecule having a function different from that of the protein, it can be applied to an optical separation column having different performance from the conventional one.

  Moreover, the bioreactor which performs two types of enzyme reaction simultaneously is obtained by combining two types of enzymes as a protein and a molecule | numerator which has a function different from this protein. For example, by combining D-aminoacylase and N-acylamino acid racemase, application to a bioreactor capable of preparing D-amino acid from N-acyl-DL-amino acid in one step is possible.

In addition, an affinity trap reactor column having the functions of a bioreactor and an affinity column can be obtained by combining an enzyme as a protein and a molecule that specifically binds to an enzyme substrate as a molecule having a function different from that of the protein. The molecule that specifically binds to the enzyme substrate is a molecule that specifically and reversibly binds to the enzyme substrate by a low-molecular ligand or antigen-antibody reaction, and can be any molecule that can bind to a porous carrier. Any one can be used as necessary. For example, by selecting and binding molecules that specifically bind to the enzyme substrate and the desired reaction product but not to the by-product, the by-product becomes an affinity trap reactor after the enzyme reaction. It is easily removed from the column and only the target product can be recovered with high purity. The following are examples of enzymes, substrates, and molecules that specifically bind to them in the affinity trap reactor.

  As shown above, when the enzyme to be bound to the porous carrier is Bacilolysin MA which is an enzyme produced by Bacillus megaterium A9542 strain, lysine (Lys) can be preferably used as such a molecule. In this case, lysine specifically binds to the substrate plasminogen and the target degradation product BL-angiostatin, but does not bind to the by-product miniplasminogen. For this reason, after the enzymatic reaction, the by-product is removed from the affinity trap reactor column without being bound to the carrier, so that only the target product can be selectively recovered.

  Examples of the carrier that can be used in the immobilization method of the present invention include a porous carrier that is usually used in affinity chromatography, depending on the type and nature of the enzyme to be immobilized and the molecule that specifically binds to the substrate. Can be selected as appropriate.

  In addition, the enzyme may be directly bonded to the porous carrier, but may be bonded via a spacer group as necessary. For example, when the porous carrier and the enzyme cannot be coupled by a direct chemical bond, or when the enzyme is a relatively small molecule and it is difficult to completely bind the enzyme and the substrate. A spacer group can be used. Such a spacer group can be appropriately selected depending on the type of enzyme to be bound.

  On the other hand, when basilolysin MA is bonded to the porous carrier, it can be easily bonded to the carrier without using a spacer.

Examples of carriers, enzymes, substrates thereof, molecules that can specifically bind to the substrate, and spacer groups that can be used preferably for the immobilization method of the present invention are listed below. However, it is not limited to these.

Example of production of affinity trap reactor by immobilization method of the present invention The affinity trap reactor according to the immobilization method of the present invention is a porous carrier in which an enzyme and a molecule that specifically binds to the substrate of the enzyme are bound. A person skilled in the art who knows how to produce affinity chromatography for use in the field of biochemistry can make it.

  In production, a spacer to be bound is selected according to the enzyme to be immobilized. When bonding the spacer, the carrier and the spacer can be easily bonded by activating the functional group of the porous carrier as necessary. For example, when PVA (polyvinyl alcohol polymer) is used as the porous carrier, the PVA carrier is epoxy-activated with epichlorohydrin and reacted with 6-aminocaproic acid to immobilize 6-aminocaproic acid as a spacer. The obtained PVA carrier. This 6-aminocaproic acid-immobilized PVA carrier can also be used as a carrier to which a molecule that specifically binds to an enzyme substrate is bound.

  Next, the enzyme is bound to the carrier to be used. In binding, the carrier and the enzyme can be easily bound by activating the functional group of the carrier as necessary. For example, when a PVA carrier to which a spacer is not bound is used as a carrier, an enzyme can be easily bound by using a CNBr activated PVA carrier activated by cyanogen bromide. In addition, when a 6-aminocaproic acid-immobilized PVA carrier is used as a carrier, NHS activity obtained by dehydrating condensation of the terminal carboxylic acid with N-hydroxysuccinimide and converting it to an N-hydroxysuccinic acid ester (NHS) group Enzyme can be easily bound by using a PVA carrier. It should be noted that the NHS activated carrier has a higher reaction rate with the enzyme and is more excellent in immobilization ability. Therefore, it is desirable to use the NHS activated carrier.

  If necessary, the activated carrier is washed with a buffer, and then the carrier is treated with an enzyme solution dissolved in the same buffer to bind the enzyme to the carrier. The concentration of the enzyme contained in the buffer can be appropriately determined according to the type of enzyme to be immobilized. In addition, the composition, pH, reaction time, and the like of the buffer solution used here can be appropriately determined according to the type of enzyme to be immobilized. For example, when immobilizing basilolysin MA on an NHS-activated PVA support, a buffer solution (composition: 0.2 M sodium bicarbonate, further about 0.01 to 1 mg / mL, preferably about 0.02 mg / mL) is used. 0.5M NaCl, containing about 5% isopropyl alcohol), and the reaction is carried out at 0 to 30 ° C. for 1 to 30 minutes, preferably at 5 ° C. for 10 minutes.

  Next, a solution of a molecule that specifically binds to the enzyme substrate is added without removing the enzyme solution, and the enzyme and a molecule that specifically binds to the enzyme substrate are bound to the carrier. The type of molecule that specifically binds to the enzyme substrate can be appropriately selected according to the type of enzyme to be immobilized. In the case of an affinity trap reactor that binds basilolysin MA, a lysine solution (composition: 2M Using a lysine solution (pH 7-8) containing about 5% isopropyl alcohol, the reaction is carried out at 0-30 ° C. for 1-240 minutes, preferably at 5 ° C. for 210 minutes.

Next, the solution of the molecule that specifically binds to the enzyme and the substrate of the enzyme is removed by filtration or the like, and then the carrier to which the enzyme and the molecule that specifically binds to the substrate of the enzyme are bound is washed with a buffer. Thus, an affinity trap reactor is obtained. The type of buffer can be appropriately selected according to the type of enzyme to be immobilized. In the case of a reactor to which basilolysin MA and lysine are bound, buffer B (composition: 25 mM sodium phosphate buffer (pH 7. 4), containing about 5% isopropyl alcohol) and the like.
The concentration of isopropyl alcohol contained in the buffer solution or aqueous solution used in each step of producing the affinity trap reactor to which the above basilolysin MA is bound is about 1 to 10%, preferably about 5%. By performing each of the above steps in the presence of isopropyl alcohol, it is possible to prevent the enzyme immobilized on the carrier from being deactivated and to keep the enzyme stably for a long period of time.

  The affinity trap reactor thus obtained is stored in a buffer at a low temperature of about −90 to 4 ° C., preferably about −10 ° C. The kind of buffer solution can be selected as appropriate. In the case of an affinity trap reactor to which basilolysin MA is bound, it can be stored in the aforementioned buffer B. By thus storing the buffer B at a low temperature, the affinity trap reactor can be stored stably over a long period of time without inactivating the enzyme.

By making the affinity trap reactor configured as described above, the reaction between the enzyme and the substrate is highly efficient, without being limited by the type of molecule to be immobilized and the enzyme that specifically binds to the enzyme substrate. And it can proceed specifically.

Example of use of affinity trap reactor according to the immobilization method of the present invention In order to proceed the reaction between a desired enzyme and a substrate using the affinity trap reactor according to the immobilization method of the present invention, the affinity trap reactor prepared as described above is used. Equilibrate in advance with buffer. The type of the buffer can be appropriately selected according to the type of the molecule that specifically binds to the immobilized enzyme and the substrate. For example, in the case of an affinity trap reactor in which basilolysin MA is bound as an enzyme and 6-aminocaproic acid is bound as a molecule that specifically binds to a substrate, 50 mM sodium phosphate buffer (pH 7.4) (buffer C) may be used. it can.

  Next, a supernatant is obtained from a sample (such as blood) such as a biological sample containing the enzyme substrate immobilized on the affinity trap reactor column by a process such as centrifugation. In the case of an affinity trap reactor in which basilolysin MA is bound as an enzyme and 6-aminocaproic acid is bound as a molecule that specifically binds to a substrate, it is treated under the conditions of centrifugation appropriately determined according to the type of sample. The enzyme is added to the affinity trap reactor that has been equilibrated to advance the reaction between the substrate and the enzyme in the biological sample, and then the affinity trap reactor is washed with buffer and then the eluate is added to the enzyme. The product produced by the reaction is eluted. As the buffer used for washing, the buffer used for equilibration can be used. For example, in the case of an affinity trap reactor in which basilolysin MA is bound as an enzyme and 6-aminocaproic acid is bound as a molecule that specifically binds to a substrate, washing is performed with the above-described buffer C containing about 0.5 M NaCl. The eluate can also be appropriately determined according to the type of enzyme and substrate. For example, in the case of an affinity trap reactor in which basilolysin MA is bound as an enzyme and 6-aminocaproic acid is bound as a molecule that specifically binds to a substrate, about 200 mM 6-aminocaproic acid is used as an eluent.

  In the case of an affinity trap reactor in which basilolysin MA is bound as an enzyme and 6-aminocaproic acid is bound as a molecule that specifically binds to a substrate, the above series of reactions are all performed at a temperature of about 0 to 50 ° C., preferably about 20 Performed at a temperature of ~ 30 ° C. By allowing the reaction to proceed at such a temperature, the basilolysin MA immobilized in the affinity trap reactor is stably maintained without self-digestion, while the action of basilolysin MA on plasminogen is ensured to the minimum necessary. Therefore, the enzyme reaction proceeds smoothly and specifically. Further, the various buffers used in this series of reactions do not contain calcium ions, and each reaction is allowed to proceed under the condition where calcium ions are not present.

  As mentioned above, although the preparation example of the affinity trap reactor by the immobilization method of this invention and its usage example were described, this invention uses BL-angiostatin from the plasminogen contained in the biological sample using this affinity trap reactor. It also relates to the method obtained in one step. In this method, a biological sample containing plasminogen is subjected to an affinity trap reactor composed of a carrier to which basilolysin MA and 6-aminocaproic acid are bound, and at a temperature of about 0 to 50 ° C., preferably about 20 to 30 ° C. By reacting in the absence of calcium ions, BL-angiostatin is obtained. 6-aminocaproic acid specifically binds both the substrate plasminogen and the target degradation product BL-angiostatin, but does not bind the byproduct miniplasminogen. . For this reason, after the enzymatic reaction, the by-product is removed from the affinity trap reactor without being bound to the carrier via 6-aminocaproic acid, so that only the target product can be selectively recovered. By proceeding the enzyme reaction using the above-described affinity trap reactor under such specific conditions, highly pure BL-angiostatin can be obtained from plasminogen in one step.

The present invention uses the affinity trap reactor obtained by the preparation example of the affinity trap reactor by the immobilization method of the present invention described in detail above, and uses the affinity trap reactor by the immobilization method of the present invention described above. Can be implemented. Specific and detailed conditions for obtaining BL-angiostatin from plasminogen in one step are as described in the following examples, and can be carried out based on the description of the examples.

The present invention will be specifically described below, but the present invention is not limited to those described in the examples.
Example 1
Preparation of PVA (polyvinyl alcohol polymerization) carrier The PVA (polyvinyl alcohol polymerization) carrier was prepared with reference to the examples described in JP-A-2007-131668.

Preparation of Epoxy Activated PVA Carrier To 249 g of PVA carrier, 400 mL of dimethyl sulfoxide (DMSO), 33.4 mL of 30% NaOH, and 267 mL of epichlorohydrin were added and reacted at 40 ° C. for 5 hours. This slurry was washed with 1.0 L of DMSO and 5.0 L of water to obtain an epoxy-activated PVA carrier.

Preparation of 6-aminocaproic acid-immobilized PVA carrier To 270 mL of epoxy-activated PVA carrier, 153 mL of water, 67.5 mL of 2M aqueous sodium carbonate solution and 49.4 mL of 2M aqueous aminocaproic acid solution were added and reacted at 50 ° C for 5 hours. The slurry was washed with 5.4 L of water to obtain an aminocaproic acid immobilized carrier.

Preparation of NHS-activated PVA carrier 97 mL of 6-aminocaproic acid-immobilized PVA carrier was washed with 243 mL of DMSO, 194 mL of DMSO, 14.3 g of NHS, and 23.8 g of EDC were added, and reacted at 40 ° C. for 10 hours. This slurry was washed with 1.9 L of DMSO with 290 mL of IPA to obtain an NHS activated PVA support.

Preparation of affinity trap reactor with immobilized basilolysin MA and lysine 10 mL of NHS-activated PVA support was washed with 50 mL of cold 1 mM HCl solution, and the washed support contained 8.81 mL of 1 mM HCl solution and then 0.4 mg of basilolysin MA 11.19 mL of buffer A (a solution containing 0.2 M sodium bicarbonate, 0.5 M NaCl, 5% isopropyl alcohol, combined with 1 mM HCl solution) was added, and the mixture was stirred at 5 ° C. for 10 minutes. Next, 2 mL of 1M lysine solution (containing 5% isopropyl alcohol) was added, and the mixture was stirred at 5 ° C. for 3.5 hours to carry out an immobilization reaction. After completion of the reaction, the solution was removed, and the gel was washed with 100 mL of buffer B (25 mM sodium phosphate containing 5% isopropyl alcohol), and finally stored at −10 ° C. or lower in buffer B having the above composition.

Preparation of affinity trap reactor column 1 mL of the affinity trap reactor support was weighed, packed in a 1 mL volume column, and stored in the aforementioned buffer B at -10 ° C or lower.

One-step purification process of BL-angiostatin from human plasma using an affinity trap reactor column An affinity trap reactor column (1 mL) was equilibrated with buffer C (50 mM sodium phosphate, pH 7.4). To the equilibrated reactor, 5 mL of citrated human plasma was added at a flow rate of 0.125 mL / min. Thereafter, the reactor was washed with 10 mL of Buffer C having the above composition containing 0.5 M NaCl (3 mL / min). The produced BL-angiostatin was eluted with 3 mL of 200 mM 6-aminohexanoic acid solution. As a result, the yield of BL-angiostatin was 163 μg.
(Comparative Example 1)
One-step purification process of BL-angiostatin from human plasma using a reactor whose purification efficiency has been reduced due to immobilization reaction due to scale-up About 65 mL of NHS-activated PVA carrier prepared according to Example 1 is cooled with 1 mM HCl solution. After washing with 1950 mL, 130 mL of Buffer A (solution containing 0.2 M sodium bicarbonate, 0.5 M NaCl, 5% isopropyl alcohol) containing 2.6 mg of basilolysin MA was added to the washed carrier, and 2 at 25 ° C. The enzyme immobilization reaction was performed by stirring for a period of time. After completion of the reaction, the reaction solution was removed, 130 mL of 0.2 M lysine solution (containing 5% isopropyl alcohol) was added, and the mixture was stirred at 25 ° C. for 2 hours to carry out lysine immobilization reaction. After completion of the reaction, the solution was removed, the gel was washed with 1950 mL of buffer B (25 mM sodium phosphate containing 5% isopropyl alcohol), and finally stored at −10 ° C. or lower in buffer B having the above composition. A column was prepared and evaluated in the same manner as in Example 1 except that the reactor shown on the left was used. As a result, the yield of BL-angiostatin was 9 μg.

Claims (4)

A method for synthesizing a column filler, comprising immobilizing a protein and a molecule having a function different from that of the protein on a porous carrier in a continuous one-pot reaction. The synthesis method according to claim 1, wherein two or more types of proteins and molecules having functions different from the proteins are combined and immobilized on the porous carrier. The synthesis according to claim 1 or 2, wherein a molecule having an affinity for an enzyme as a protein and a molecule having a function different from that of the protein is affinity to a substrate of the enzyme or a degradation product of the substrate by the enzyme. Method. Vasilolysin MA, which is a protease using plasminogen as a substrate as an enzyme, and affinity for plasminogen degradation product by plasminogen or basilolysin MA as a molecule having affinity with the enzyme substrate or enzyme degradation product of the substrate. The synthesis method according to any one of claims 1 to 3, wherein lysine or / and 6-aminocaproic acid, which is a molecule possessed, is immobilized.