JPH05336986A - Protein-reactivating agent - Google Patents

Abstract

PURPOSE:To provide the protein-reactivating agent free from physiological activity due to its steric structure. CONSTITUTION:The protein-reactivating agent having the same primary structure as a protein having a physiological activity but not having a physiological activity because of its having a different steric structure contains DsbA as an active ingredient. The DsbA (another name: PpfA) is an enzyme known to be present in the periplasm fraction of Escherichia coli. A protein free from physiological activity can be reactivated in a short time without considerably increasing the volume of a protein solution.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a protein reactivating agent, and more specifically, it is a protein produced by a microorganism based on, for example, a genetic engineering method, and its three-dimensional structure is different from that of a natural protein. Therefore, the present invention relates to a reactivating agent for a protein having no physiological activity due to its three-dimensional structure, such as a protein having no physiological activity.

[0002]

2. Description of the Related Art Recombinant DNA technology has made it possible to prepare a large amount of a substance such as a peptide or protein of biological origin, which was extremely difficult to isolate as a trace component, by using a microorganism. .. However, it is known that heterologous proteins expressed using bacteria and the like often exist in a biologically inactive state as a denatured precipitate in host cells. It is considered that this is because the primary structure of the protein is a natural type correct structure derived from recombinant DNA, but its three-dimensional structure is not a biologically correct specific structure. .. Also,
A chemically synthesized polypeptide or a protein synthesized in a cell extraction solution often does not have a correct three-dimensional structure, lacks a disulfide bond, and often has an inactive structure with relatively few folds. As a method for recovering the activity of a protein having no physiological activity due to such a three-dimensional structure, JP-A-59-161321 discloses that an inactive precipitated protein is solubilized using a strong denaturant. Then
It is disclosed that the solubilized solution is diluted or replaced with a weak denaturing solution to recover the active form. However, such a method has the drawback that it results in a significant increase in the volume of the protein solution and that it takes a significantly longer time to recover the activity.

[0003]

The object of the present invention is a reactivating agent for a protein having no physiological activity due to its three-dimensional structure, which can be carried out in a shorter time than conventional methods. Moreover, it is to provide a reactivating agent that can be carried out without increasing the volume of the protein solution so much.

[0004]

SUMMARY OF THE INVENTION The present invention can be summarized as follows: The present invention reactivates a protein having the same primary structure as a physiologically active protein, but having no three-dimensional structure and therefore having no physiological activity. The agent is characterized by containing DsbA as an active ingredient.

The present invention will be described in detail below. DsbA (also known as PpfA) used in the present invention is an enzyme found to be present in the periplasmic fraction of Escherichia coli, and has been reported to catalyze the reduction of oxidized insulin in the presence of sulfhydryl compounds. [The Emboss
Journal (The EMBO Journal), Volume 11, 55
-62 (1992) and Cell, Vol. 67, 581-589 (1991)]. DsbA may be prepared from the periplasmic fraction of Escherichia coli, or may be used by expressing DsbA in a large amount by a genetic engineering technique. For example, Escherichia coli containing the plasmid pKY220 described in the above-mentioned Diembo Journal.
The target protein expressed by HB101 / pKY220) may be purified and then used. For example, the strain is cultured in an LB medium at 37 ° C. until the rate log phase, the cultured bacterial cells are collected by centrifugation, and then the bacterial strain is subjected to an ice-cooled osmotic shock method [Journal of Biological
Chemistry (J. Biol. Chem.), Volume 240, Volume 36
65-3692 (1965)], and then the fraction is dialyzed against, for example, 10 mM Tris-HCl buffer (pH 8.1), followed by dialysis with DEAE-. After passing through a Sepharose CL-6B column and re-dialyzing the non-adsorbed fraction, 0-0.3 M KCl
Column chromatography on a linear concentration gradient DEAE-Sepharose CL-6B column of 0.15M
A DsbA solution may be prepared as a KCl elution fraction.

The effective amount of DsbA used in the present invention is
Examples of the protein reactivating agent containing:
A preparation containing an effective amount of purified DsbA, which may be in the form of solution or lyophilized product. The substance used in the formulation may be any substance as long as it can be used for ordinary enzyme preparations.

The protein that can be reactivated by the reactivating agent of the present invention has the same primary structure as that of the active protein, but the three-dimensional structure of the protein is active because the disulfide bond is not normal. Any protein may be used as long as it has no physiological activity because it has a different structure. As an example of such a thing, various polypeptides chemically synthesized by a peptide synthesizer, Escherichia coli S
Transcription / translation coupled system using 30 extracts [Annual Review of Ge
netics), vol. 7, pp. 267-287 (197)
3)], or a translation system using wheat germ or rabbit reticulocyte extract [Methods in Enzymology, Vol. 101, 635]
Pp. 644 (1983), and Canadian Journal of Biochemistry and Cell B.
iology), 61, 274-286 (198).
3)] and various proteins and polypeptides produced by microorganisms by the recombinant DNA method, such as urokinase, prourokinase, insulin, somatomedin C, tissue plasminogen activator, hybrid plasminose Examples thereof include gen activator, interferon, calcitonin, proteins derived from hepatitis B virus and poliovirus, and lymphokines such as lymphotoxin, γ-interferon, interleukin 2 and granulocyte macrophage colony stimulating factor.

When reactivating an inactive protein with the reactivating agent of the present invention, the amount of DsbA is not particularly limited.
As a mode of acting DsbA on a protein, a solution in which DsbA is dissolved in a buffer may be added to the protein, or after solubilizing the protein with an appropriate denaturant,
DsbA may be added to the protein solution. The reaction temperature is preferably 0 to 40 ° C., and the reaction time is appropriately 1 to 5 hours.

As mentioned above, D
Although it is possible to directly act on sbA, in the case of an insoluble protein, the protein is first dissolved in a reducing denaturing solution, and then Dsb is added in the presence of a disulfide compound.
It is preferable to act A. The reduction denaturing solution is preferably 0.1-0.2 M dithiothreitol, 1-6 M guanidine hydrochloride solution, or 1-8 M urea solution,
The concentration of inactive protein dissolved in the denaturing solution is 0.
About 1 to 40 mg / ml is suitable. Further, as the disulfide compound, oxidized glutathione or oxidized dithiothreitol is preferable. The disulfide compound should be added at a concentration higher than the molar concentration of the sulfhydryl compound in the entire reaction solution.

[0010]

EXAMPLES Examples of the present invention will be given below, but the present invention is not limited to these examples.

Example 1 Preparation of DsbA Pp containing a plasmid pKY220 containing a gene encoding PpfA described in the above-mentioned Diemvo Journal.
fA overproducing E. coli K12 strain (Escherichia coli HB
101 / pKY220) was cultured in LB medium at 37 ° C. until the rate log phase, and the cultured cells were collected by centrifugation. Next, a periplasmic fraction was obtained from the obtained bacterial cells by using the ice-cooled osmotic shock method described above, and the fraction was diluted with 10 mM Tris-hydrochloric acid buffer (pH 8.
1), dialyzed solution was passed through a DEAE-Sepharose CL-6B column, and the non-adsorbed fraction was redialed, and then a linear gradient DEAE-Sepharose CL-6B column of 0 to 0.3 M KCl was used. Column chromatography was performed at 0.1 to prepare a 0.15 M KCl elution fraction as a DsbA-containing solution.

Example 2 (1) Synthesis of Inactive Alkaline Phosphatase Plasmid pBA74 containing a gene encoding alkaline phosphatase produced by Baba et al. [Journal of Bacteriol]
Ogy), Volume 172, 7005-7010 (199
0)], [ ³⁵ S] methionine-containing Escherichia coli S30 transcription / translation coupling system [Annual Review of Genetics, Vol. 7, pp. 267-287 (19).
73)] was used to synthesize alkaline phosphatase. Protein synthesis was stopped by adding chloramphenicol at a final concentration of 100 μg / ml, of which 5 μl
To a final volume of 16.1 μl with 0.16 mM Zn
An insoluble alkaline phosphatase solution was prepared by diluting with 0.47 M Tris acetate buffer containing Cl ₂ .

(2) D of inactive alkaline phosphatase
Reactivation by sbA To the solution containing the alkaline phosphatase obtained in Example 2- (1), oxidized glutathione and DsbA were added, and the mixture was kept warm at 37 ° C and then activated from the inactive reduced alkaline phosphatase. The conversion to the native form of oxidized alkaline phosphatase was tested over time. Example 2-
Since the reduced alkaline phosphatase synthesized in the cell-free system of (1) lacks a disulfide bond, it has few folds in the three-dimensional structure of the whole protein, is easily digested by trypsin, and has no enzymatic activity. But,
Once the sulfhydryl group is oxidized and disulfide formation occurs, the overall three-dimensional structure changes, resistance to trypsin digestion occurs, the enzyme forms a dimer, and the enzyme activity is activated.

The presence or absence of a disulfide bond in the monomeric alkaline phosphatase was detected by the difference in the migration distance of the protein in non-reducing SDS polyacrylamide gel electrophoresis. The conversion from reduced alkaline phosphatase to oxidized alkaline phosphatase is shown in FIG. 1 as a relationship between time (minutes, horizontal axis) and oxidized active enzyme (%, vertical axis). In Fig. 1, white circles indicate 0.07 in the alkaline phosphatase solution after synthesis.
The results when 5 mM oxidized glutathione was added are shown by black circles: 0.075 mM oxidized glutathione and 0.55 mg.
/ Ml DsbA was added, the black triangle indicates 0.
075 mM oxidized glutathione and 1.1 mg / ml Ds
The results when bA is added are shown. As shown in FIG. 1, when DsbA was added, the appearance of oxidized alkaline phosphatase was promoted, the appearance frequency thereof depended on the DsbA concentration, and the activation of the enzyme activity also depended on the DsbA concentration.

Example 3 (1) Ribonuclease reduction modification treatment Bovine ribonuclease (manufactured by Sigma) was used at a final concentration of 40.
120 mM dithiothreitol at 2 mg / ml
The enzyme was suspended in 50 mM Tris-hydrochloric acid buffer solution (pH 8.1) containing mM EDTA and 6 M guanidine hydrochloride and kept at 24 ° C. for 20 hours to denature the enzyme. By this process,
The residual activity of ribonuclease was 3.3%. Using this inactivated ribonuclease solution, activation with DsbA was performed.

(2) Activation of Enzyme by DsbA Treatment DsbA treatment was carried out by the following two methods. Method I: 0.55 mg / inactivation ribonuclease solution
45 ml of DsbA, 0.9 mM oxidized glutathione
A volume of 1/200 was added to mM Tris acetate buffer (pH 8.1), the mixture was kept at 37 ° C. and the ribonuclease activity was measured over time. Method II: Inactivating ribonuclease solution was adjusted to pH 3.0 with 1N HCl and 0.2M NaC was added.
Sephadex G-2 swollen with 10 mM HCl
After passing through 5 columns, dithiothreitol and guanidine hydrochloride were removed, and purified reduced DsbA was recovered.
Next, 0.6 mM dithiothreitol, 0.9 mM oxidized glutathione, 1 mM MgCl ₂ , 0.55 mg / ml Ds was added so that the ribonuclease had a final concentration of 0.2 mg / ml.
45 mM trisacetate buffer (pH 8.
1), the mixture was kept warm at 37 ° C., and the ribonuclease activity was measured over time.

The activity of the ribonuclease treated by Method I or Method II is shown in FIG. 2 as a relationship between time (minutes, horizontal axis) and ribonuclease activity (% to equivalent amount of active enzyme, vertical axis). The ribonuclease activity was measured by the Blackburn method [Journal of Biological Chemistry, Vol. 254, No. 12484-12.
Pp. 487 (1979)]. The black circles in FIG. 2 show the results when DsbA was added in Method I, the white circles show the results when Method I was not added, and the black triangles in FIG. 2 show the results when DsbA was added in Method II. The result is the white triangle marked Method II
Shows the result in the case of no addition. As shown in FIG.
DsbA activates a remarkable enzyme activity of inactivated ribonuclease.

[0018]

EFFECT OF THE INVENTION By using the reactivating agent of the present invention, a protein having no physiological activity due to its three-dimensional structure different from that of the natural type is effectively reactivated in a shorter time than the conventional method. can do. Also, compared to the conventional method,
Much less increase in protein solution volume. According to the present invention, a protein synthesized based on a biochemical or genetic engineering method and further purified, which has no physiological activity due to its three-dimensional structure different from that of the natural type, can be efficiently reactivated within a short time. As such, the present invention is believed to provide significant benefits to the commercial practice of active protein manufacturing.

[Brief description of drawings]

FIG. 1 is a diagram showing the conversion of reduced alkaline phosphatase into oxidized alkaline phosphatase by DsbA.

FIG. 2 is a diagram showing the activity recovery of inactivated ribonuclease by DsbA.

Front page continuation (72) Inventor Noriko Kusunagawa 3-4-1 Seta, Otsu City, Shiga Prefecture, Central Research Institute, Minami Shuzo Co., Ltd. (72) Ikunobu Kato 3-4-1 Seta, Otsu City, Shiga Prefecture Sake Brewing Co., Ltd. Central Research Institute

Claims (1)

[Claims] 1. A reactivating agent for a protein which has the same primary structure as a physiologically active protein but does not have a physiological activity due to a different three-dimensional structure, and which comprises Ds
A protein reactivating agent comprising bA as an active ingredient.