KR100247151B1 - A refolding system for recombinant proteins expressed in escherichia coli as inclusion bodies - Google Patents

Abstract

The present invention relates to a system that can quickly perform the unwinding and reconstitution of the recombinant protein inclusion body under various conditions by various unwinding conditions and reconstitution conditions. The unwinding and reconstitution system of the recombinant protein inclusion body of the present invention is a buffer for carrying out the unwinding process under a distilled water supply unit, an inclusion body suspension for dissolving the recombinant protein inclusion body introduced into the unwinding and reconstitution process, a plate and m conditions. A releasing unit including an annealing reagent consisting of a solution component and an additive, a reconstituting unit including a reagent for reconstituting a plate and a mixture of a buffer component, an additive, and an oxidation / reducing agent capable of performing a reconstitution process under n conditions, It includes a loading tray unit, a reaction unit for reacting the plate undergoing the reconstruction process, and a reconstruction measuring unit for confirming the degree of reconstitution of the reconstituted protein. When the recombinant protein inclusion body is solved and reconstituted by the method of the present invention, it is possible to quickly perform various unfolding and reconstitution of a total of m × n kinds, thereby accurately setting reconstitution conditions capable of restoring the activity of the target protein most intactly.

Description

Unwinding and Reconstitution System of Recombinant Protein Inclusion Body

The present invention relates to an automated system for unfolding and refolding a recombinant protein inclusion body. More specifically, the present invention relates to a system capable of quickly performing annealing and reconstitution of a recombinant protein inclusion body under various conditions by combining various annealing conditions and reconstitution conditions.

In a physiological environment, the structure of a protein is stabilized in a complex form as a result of interactions between the protein itself and its entropy relationship. The stabilizing and destabilizing forces of these proteins are balanced. Therefore, even a slight change in the surrounding environment can easily destabilize the structure of the protein. As such, the formation and maintenance of a specific protein in its physiological environment can be seen as the result of constant interaction with the properties of the protein itself and the surrounding environment.

Recently, with the development of genetic recombination technology, there have been many attempts to produce a large amount of proteins useful to humans using E. coli, and related technologies are rapidly developing. However, when the recombinant protein is produced by genetic recombination technology, when such protein is overexpressed, it frequently denatures without inherent reconstitution process, entangles with each other, and forms a large protein mass called an inclusion body. . Since these inclusion bodies are irreversibly denatured proteins that cannot return to their intrinsic structure under normal physiological conditions, there is a problem that they do not exhibit the expected activity of the target protein (see Jaenicke, R. and Rudolph, R., Meth). Enzymol., 131: 218-250 (1986); Jaenicke, R., Prog.Biophys.Molec.Biol., 49: 117-237 (1987); Rudolph, R. et al., Biochemistry, 18: 5572- 5575 (1979); Zettlmeissl, G. et al., Eur. J. Biochem., 121: 169-175 (1981); Anfinsen, CB, Angrew. Chem., 85: 1065-1074 (1973).

In order to solve this problem, there have been many studies on the annealing process of releasing the protein entangled in the inclusion body and the process of reconstituting the released protein into its own tertiary structure. As an annealing process to release the protein in the inclusion body, a method of using a protein denaturant such as urea or guanidine hydrochloric acid (guanidine HCl) at a high concentration of 6 to 10 M or adjusting the pH with a high concentration of sodium hydroxide and hydrochloric acid has been proposed. . In addition, research on reconstitution has also been carried out to reduce the type and pH of buffers, protein concentrations, types and concentrations of oxidation / reducing agents, methods of lowering protein denaturant concentrations, protein structure stabilizers such as glycerol, ethylene glycol, sucrose, and the like. Various variables have been proposed for consideration of reconstitution of proteins, including the use of additives such as cations or anions, and the addition of protease inhibitors (see Winfield, PT et al., Current Protocol in Protein Science, Vol. 1: 6.5). .1-6.5.27, Wiley and Sons, Inc., New York.

However, it takes a long time to perform the annealing and reconstitution of a protein in which such various variables work, for example, one researcher unpacks and reconstructs the inclusion body by a combination of 200 to 300 kinds a week. In this case, it takes about 3 to 4 months to try all 3,000 types of cases, and considerable time and personnel are required to set the conditions for reconstructing the denatured protein in the inclusion body into its own structure. It is necessary. Accordingly, since most of the recombinant proteins produced as a result of uninterrupted efforts by the genetic recombination technology do not have activity in the inclusion body state, it is possible to promptly condition the conditions for unwinding and reconstructing the structure so that the desired recombinant protein exhibits unique activity. It was urgent to develop a technology that can be set up.

Accordingly, the present inventors have made extensive efforts to select and combine various unwinding and reconstitution conditions of E. coli-derived recombinant protein to develop a system for quickly setting the unwinding and reconstituting conditions of a recombinant protein inclusion body. By unpacking and reconstructing the inclusion body by the combination, it was confirmed that the correct loosening and reconstitution conditions can be set quickly, and the present invention was completed.

After all, it is an object of the present invention to provide a system capable of quickly setting the unwinding and reconstitution conditions of a recombinant protein inclusion body.

Figure 1 is a schematic diagram showing the configuration of the unwinding and reconstitution of the recombinant protein inclusion body according to an embodiment of the present invention.

In the present specification, the unwinding and reconstitution system of the recombinant protein inclusion body of the present invention includes unfolding and eight kinds of additives and three kinds of buffer components by the combination of two kinds of buffer components and six kinds of additives. For example, a case of unwinding and reconstructing a recombinant protein inclusion body under a total of 3,456 conditions by refolding by 288 combinations of four kinds of pH conditions and three kinds of oxidation / reduction conditions is given. However, if necessary, the number of conditions can be reduced or expanded since the researcher can easily modify the type and additives of the buffer used in the system.

Hereinafter, the unwinding and reconstitution system of the recombinant protein inclusion body according to the present invention will be described in detail.

Unwinding and reconstitution system of the recombinant protein inclusion body of the present invention,

Distilled water supply unit;

Inclusion body suspension for dissolving the recombinant protein inclusion body to be introduced during annealing and reconstitution;

An unpacking unit including a unpacking reagent including a plate and a buffer component and an additive capable of performing an unwinding process under m conditions;

A reconstruction unit including a reconstituting reagent composed of a mixture of a plate, a buffer component, an additive, and an oxidation / reducing agent capable of performing a reconstruction process under n conditions;

A reconstruction plate mounting portion for loading a plate to be used for reconstruction;

A reaction unit for reacting the plate in which the reconstruction process is in progress; And,

Reconstruction measurement unit for confirming the degree of reconstitution of the reconstituted protein is included.

In this case, the annealing reagent may include 5 to 8 M, most preferably 7 M of guanidine hydrochloride (guanidine HCl); Tris or phosphate from 10 to 100 mM, most preferably 50 mM; Guanidine hydrochloric acid buffer solution (pH 6.0 to 11.0) comprising 0.5 to 100 mM, most preferably 1 mM EDTA; 5 to 9 M, most preferably 8 M of urea; 10 to 100 mM, most preferably 50 mM of tris or phosphate; Urea buffer (pH 6.0 to 11.0) comprising 0.5 to 100 mM, most preferably 1 mM EDTA; Dithiothretol or dithioerythritol of 1 to 50 mM, most preferably 5 mM; 50 to 250 mM, most preferably 50 mM of dithiothreitol or dithioerythritol; Sodium sulfite at 0.05-2.0 mg / ml, most preferably 0.2 mg / ml; Sodium sulfite at 1 to 5 mg / ml, most preferably 2 mg / ml; Sodium sulfite from 10 to 50 mg / ml, most preferably 20 mg / ml; Sodium tetrathionate at 0.01 to 1.0 mg / ml, most preferably 0.1 mg / ml; 0.5 to 2.5 mg / ml, most preferably 1 mg / ml of sodium tetrathionate; And 5 to 25 mg / ml, most preferably 10 mg / ml of sodium tetrathionate. Thus, the annealing process is a combination of two cases by the electric buffer component, two cases by the concentration of dithiothritol or dithioerythritol, and three cases by the addition amount of sodium sulfite and sodium tetrathionate. Proceed to 12 cases. At this time, the concentration of the recombinant protein is 0.1-100mg / ml, most preferably 1-20mg / ml.

In addition, the reagent for reconstitution includes 0.1 to 1 M, most preferably 0.5 M of L-arginine, 0.05 to 1%, most preferably 0.01% of tween 80, 0.01 to 1 %, Most preferably 0.02% tween 20, 1-20%, most preferably 5% sucrose, 0.1-1%, most preferably 0.2% mannose and lactose mixture, 0.1 To 5%, most preferably 1% mannitol, 0.05-1%, most preferably 0.1% human serum albumin; And 20 to 200 mM, most preferably 50 mM Tris HCl buffer (pH 7.0, 7.5, 8.0 and 8.5), sodium phosphate buffer (pH 7.0, 7.5, 8.0 and 8.5) and glycine: 1 mM: 0.1 mM mixture of sodium hydroxide (Glycine: NaOH) buffer (pH 9.0, 9.5, 10.0 and 10.5) and glutathione oxide and reduced glutathione (GSH: GSSG) or cysteine: cysteine as oxidation / reducing agent or 10 mM: 1 mM mixtures are included. Thus, the reconstitution process consists of eight types including seven kinds of additives and nothing added, three cases based on buffer components, four cases based on pH conditions for each buffer component, and two kinds of oxidizing / reducing agent mixtures. It proceeds to the number of 288 cases by the combination of three cases by adding or not adding. The concentration of the suture protein at reconstitution is 0.005-10 mg / ml, most preferably 0.01-5 mg / ml.

In addition, if all 12 cases of the electric release process and 288 cases of the reconstruction process are combined, a total of 3,456 cases can be implemented.

On the other hand, it is preferable that the reaction unit is provided with a temperature controller and a stirring device that can adjust the temperature in the range of 4 to 50 ℃, the reconstruction unit is a light scatterer, optical density meter, fluorescence meter, CD (circular dichroism) By selecting and interconnecting an optical rotatory detector (ORD), a device for conducting SDS-PAGE, and a densitometer, the recombination status of the recombinant protein is analyzed, and only the sample showing the most suitable degree of reconstruction is taken. Final confirmation by measuring the physiological activity in the facility. On the other hand, the annealing and reconstitution reagents may be prepared in a liquid phase, or dissolved in distilled water immediately before use, and may be prepared in a kit by lyophilization to be used.

In addition, in order to facilitate the unwinding and reconstitution of the recombinant protein inclusion body, the inclusion body dissolution frame, the sample delivery trap, the loading frame and the robot arm may be further included and automated.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 setting of annealing and reconstitution conditions of E. coli-derived recombinant protein

Among the various unwinding and reconstitution conditions of the E. coli-derived recombinant protein, general conditions were selected and combined to set 12 optimal conditions for the unwinding process and 288 optimal conditions for the reconstructing process as shown in Table 1 and Table 2 below.

12 optimal conditions for loosening process Buffer Dithiothreitol (DTT) (or dithioerythritol (DTE)) Sodium sulfite (SS) + sodium tetrathionate (STT) Guanidine hydrochloride buffer (pH 6.0 ~ 11.0): EDTA with 1 mM of 50 mM Tris (or phosphate) 7M guanidine hydrochloride 5 mM 0.2mg / ml + 0.1mg / ml 2mg / ml + 1mg / ml Urea buffer solution (pH 6.0 to 11.0): 8 mM urea 50 mM Tris (or phosphate) 1 mM EDTA 50mM 20mg / ml + 10mg / ml Buffer 2 condition x DTT (or DTE) 2 condition x (SS + STT) 3 condition = 12 condition

288 optimal conditions for the reconstruction process basic Buffer pH Glutathione oxide: Reduced glutathione (or cysteine: cystine) 0.5M L-arginine 50 mM Tris buffer 7.0, 7.5, 8.0, 8.5 1 mM: 0.1 mM mixture 0.01% Tween 80 0.02% Tween 20 50 mM phosphate buffer 10mM: 1mM mixture 5% sucrose 0.2% Mannose + Lactose Mixture 50 mM Glycine: Sodium Hydroxide Buffer 9.0, 9.5, 10.0, 10.5 No addition 1% mannitol 0.1% human serum albumin No addition Basic 8 conditions x Buffer 3 conditions x pH 4 conditions x oxidation / reduction 3 conditions = 288 conditions

Twelve optimal conditions for the annealing process and 288 optimal conditions for the reconstitution process, as shown in Table 1 and Table 2, were selected and set, and when these two conditions were combined, a total of 3,456 protein reconstitutions were possible.

Example 2 Preparation of Unwinding and Reconstitution Kit Device of Genetic Protein Inclusion Body

In one embodiment of the present invention, the structure of the unwinding and reconstitution kit device of the recombinant protein inclusion body is schematically illustrated in FIG. 1.

As shown in FIG. 1, the annealing and reconstitution kit apparatus of the present invention includes distilled water and solution feeder, an inclusion suspension for dissolving inclusion bodies introduced into an annealing and reconstitution process, an annealing comprising six 96-well plates and an annealing reagent. Unfolder, three 96-well plates and a refolder containing reagents for reconstitution, a loading tray, and a thermostat with temperature controlled in the range of 4-50 ° C. for reaction of the plate undergoing the reconstruction process An optical density meter was included to measure the degree of reconstitution of the installed reactor and the reconstituted protein.

In this case, the annealing reagent includes 7M guanidine hydrochloric acid, 50mM tris or phosphate, 1mM EDTA guanidine hydrochloride buffer solution (pH 6.0 to 11.0), 8M urea, 50mM tris or phosphate, 1mM EDTA. Urea buffer solution (pH 6.0 to 11.0), 50mM dithiothreitol or dithioerythritol, 200mg / ml sodium sulfite, 100mg / ml sodium tetrathionate and distilled water was prepared in a volume of 10ml each, Reagents for reconstitution include 0.5M L-arginine, 0.01% Tween 80, 0.02% Tween 20, 5% Sucrose, 0.2% Mannose and Lactose Mixture, 1% Mannitol, 0.1% Human Serum Albumin , Tris buffer solution at 4 pH conditions (pH 7.0, 7.5, 8.0 and 8.5) at 50mM, phosphate buffer solution at 4 pH conditions (pH 7.0, 7.5, 8.0 and 8.5) at 50mM, 4 pH conditions (50mM) glycine: sodium hydroxide buffer at pH 9.0, 9.5, 10.0 and 10.5) Solution, 1 mM: 0.1 mM mixture, 10 mM: 1 mM mixture of distilled water and 10 ml of each of the glutathione oxide and reduced glutathione (GSH: GSSG) or cysteine and cysteine (cystine) were prepared.

Example 3 Unwinding and Reconstitution of Genetic Tissue Specific Plasminogen Activator (t-PA) Using Unwinding and Reconstitution of Genetic Protein Inclusion Kit Kit

In order to confirm the effect of the unwinding and reconstitution of the recombinant protein inclusion body using the unwinding and reconstitution kit apparatus of the recombinant protein inclusion body prepared in Example 2, plasminogen activator (t-PA) suture expressed from the recombinant E. coli The sieve was loosened and reconstituted under 12 conditions and 288 conditions, and the turbidity (absorbance at 400-750 nm) and protein activity of the reconstituted protein were measured by conventional methods.

Tables 3a and 3b below reconstitute the annealing samples in Tris buffer, measure turbidity, and measure activity, and each reconstitution condition is as follows: A-50 mM Tris buffer (pH 7.0) ), No oxidation / reducing agent; B-50 mM Tris buffer (pH 7.5), no oxidation / reducing agent added; C-50 mM Tris buffer (pH 8.0), no oxidation / reducing agent added; D-50 mM Tris buffer (pH 8.5), no addition of oxidation / reducing agent; E-50 mM Tris buffer (pH 7.0), addition of 1 mM: 0.1 mM mixture of glutathione oxide and reduced glutathione (GSH: GSSG); F-50 mM Tris buffer (pH 7.5), addition of 1 mM: 0.1 mM mixture of oxidized glutathione and reduced glutathione (GSH: GSSG); Addition of G-50 mM Tris buffer (pH 8.0), a 1 mM: 0.1 mM mixture of oxidized glutathione and reduced glutathione (GSH: GSSG); Addition of a 1-50 mM mixture of H-50 mM Tris buffer (pH 8.5), glutathione oxide and reduced glutathione (GSH: GSSG); I-50 mM Tris buffer (pH 7.0), addition of a 10 mM: 1 mM mixture of reduced glutathione oxide and reduced glutathione (GSH: GSSG); J-50 mM Tris buffer (pH 7.5), 10 mM: 1 mM mixture of oxidized glutathione and reduced glutathione (GSH: GSSG); K-50 mM Tris buffer (pH 8.0), addition of 10 mM: 1 mM mixture of glutathione oxide and reduced glutathione (GSH: GSSG); Addition of L-50 mM Tris buffer (pH 8.0), a 10 mM: 1 mM mixture of oxidized glutathione and reduced glutathione (GSH: GSSG); 1-no addition; 2-0.5M L-arginine; 3-0.01% tween 80; 4-0.02% Tween 20; 5-5% sucrose; 6-0.2% mannose + 0.2% lactose; 7-1% mannitol; And, 8-0.1% human serum albumin.

As shown in the table below, when the correlation between turbidity and protein activity is compared, it can be seen that the activity of t-PA is high in the samples showing low turbidity. Therefore, when unwinding and reconstitution of the t-PA inclusion body using the kit device of the present invention, when the turbidity is low, it can be seen that the t-PA was prepared to be properly reconstituted to maintain the intrinsic activity. .

Turbidity measured after reconstruction of t-PA One 2 3 4 5 6 7 8 A 0.114 0.027 0.021 0.014 0.022 0.019 0.024 0.027 B 0.131 0.027 0.018 0.015 0.018 0.026 0.028 0.024 C 0.139 0.034 0.022 0.016 0.023 0.029 0.034 0.029 D 0.124 0.029 0.018 0.008 0.021 0.029 0.018 0.019 E 0.031 0.026 0.035 0.022 0.034 0.031 0.025 0.033 F 0.028 0.022 0.014 0.017 0.019 0.022 0.034 0.026 G 0.021 0.022 0.013 0.009 0.009 0.024 0.022 0.019 H 0.018 0.025 0.011 0.008 0.013 0.015 0.027 0.014 I 0.031 0.031 0.036 0.027 0.031 0.031 0.031 0.033 J 0.019 0.028 0.021 0.017 0.022 0.023 0.023 0.025 K 0.018 0.022 0.013 0.008 0.014 0.017 0.015 0.016 L 0.029 0.016 0.006 0.001 0.004 0.001 0.001 0.005

Activity measured after reconstitution of t-PA (unit: OD ₄₀₅ ) One 2 3 4 5 6 7 8 A 0.009 0.003 0.001 0.001 0.001 0.001 0.001 0.002 B 0.016 0.006 0.002 0.001 0.001 0.001 0.001 0.003 C 0.046 0.031 0.009 0.003 0.007 0.007 0.003 0.023 D 0.131 0.071 0.035 0.017 0.032 0.025 0.023 0.164 E 0.004 0.015 0.002 0.001 0.003 0.001 0.001 0.014 F 0.005 0.028 0.007 0.002 0.003 0.005 0.007 0.034 G 0.111 0.078 0.107 0.035 0.191 0.097 0.103 0.147 H 0.501 0.168 0.248 0.285 0.469 0.395 0.371 0.407 I 0.014 0.043 0.012 0.008 0.022 0.016 0.023 0.025 J 0.049 0.062 0.035 0.014 0.061 0.051 0.064 0.079 K 0.234 0.136 0.168 0.078 0.294 0.276 0.263 0.266 L 0.485 0.217 0.405 0.304 0.512 0.542 0.501 0.386

As described and demonstrated in detail above, the present invention provides a system that can accurately and quickly perform the unwinding and reconstitution of the recombinant protein inclusion body. When the recombinant protein inclusion body is solved and reconstituted by the method of the present invention, various annealing and reconstitution can be quickly performed to accurately set conditions for reconstitution while maintaining the activity of the target protein intact.

Claims (6)

Distilled water supply unit; Inclusion body suspension for dissolving the recombinant protein inclusion body to be introduced during annealing and reconstitution; An unpacking unit including a unpacking reagent including a plate and a buffer component and an additive capable of performing an unwinding process under m conditions; A reconstruction unit including a reconstituting reagent composed of a mixture of a plate, a buffer component, an additive, and an oxidation / reducing agent capable of performing a reconstruction process under n conditions; A reconstruction plate mounting portion for loading a plate to be used for reconstruction; A reaction unit for reacting the plate in which the reconstruction process is in progress; And, An unwinding and reconstitution system of a recombinant protein inclusion body comprising a reconstitution measurer for confirming the reconstruction degree of the reconstructed protein. The method of claim 1, The reagent for annealing includes guanidine hydrochloric acid buffer solution (pH 6.0 to 11.0), 5 to 9M urea, 10 to 100 mM, including 5 to 8 M guanidine hydrochloric acid, 10 to 100 mM tris or phosphate, 0.5 to 100 mM EDTA. Tris or phosphate, urea buffer solution containing 0.5 to 100 mM EDTA (pH 6.0 to 11.0), 1 to 50 mM dithiothreitol or dithioerythritol, 50 to 250 mM dithiothreitol or dithioerythritol , 0.05-2.0 mg / ml sodium sulfite, 1-5 mg / ml sodium sulfite, 10-50 mg / ml sodium sulfite, 0.01-1.0 mg / ml sodium tetrathionate, 0.5-2.5 mg / ml Sodium tetrathionate and 5 to 25 mg / ml sodium tetrathionate, two cases by electrical buffer component, two cases by dithiothreitol or dithioerythritol concentration and The annealing process is carried out in the number of 12 cases (m) by the combination of three cases by the addition amount of sodium sulfite and sodium tetrathionate. Unwinding and reconstitution system of the recombinant protein inclusion body. The method of claim 1, Reagents for reconstitution include 0.1-1 M L-arginine, 0.05-1% Tween 80, 0.01-1% Tween 20, 1-20% Sucrose, 0.1-1% Mannose and Lactose Mixture, 0.1-5% mannitol, 0.05-1% human serum albumin, 20-200 mM Tris buffer (pH 7.0, 7.5, 8.0 and 8.5), 20-200 mM phosphate buffer (pH 7.0, 7.5, 8.0 And 8.5) and 20-200 mM glycine: sodium hydroxide buffer (pH 9.0, 9.5, 10.0 and 10.5) and 1 mM of glutathione oxide and reduced glutathione (GSH: GSSG) or cysteine and cysteine as an oxidation / reducing agent. Eight cases, including: 0.1 mM mixture or 10 mM: 1 mM mixture, including eight kinds of additives and no additives, three cases with buffer components, four cases with pH conditions for each buffer component With or without the addition of two oxidizing / reducing agent mixtures If the reconstruction process by a combination of three case by the case of 288 kinds, characterized in that the progress of the number (n) Unwinding and reconstitution system of the recombinant protein inclusion body. The method of claim 1, The reaction unit is characterized in that the temperature controller is installed to control the temperature in the range of 4 to 50 ℃ Unwinding and reconstitution system of the recombinant protein inclusion body. The method of claim 1, The reconstruction measuring unit further includes an apparatus selected from the group consisting of a light scattering device, an optical density measuring instrument, a fluorescence measuring instrument, a circular dichroism (CD), an optical rotatory detector (ORD), an SDS-PAGE, and a densitometer. Characterized in that Unwinding and reconstitution system of the recombinant protein inclusion body. The method of claim 1, And further comprising a component selected from the group consisting of an enclosure melting frame, a sample delivery trap, a loading frame, and a robotic arm. Unwinding and reconstitution system of the recombinant protein inclusion body.

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