KR980009465A - Method for producing low molecular weight heparin using recombinant heparin-degrading enzyme comprising histidine oligopeptide - Google Patents

Abstract

The optimal pH and optimal temperature of the recombinant heparin-degrading enzyme, which contains recombinant heparin degrading enzyme and histidine oligopeptide, were investigated by inserting the gene of heparin-degrading enzyme into E. coli expression vectors pET11a and pET16b from F. Both heparin degrading enzymes including recombinant heparin degrading enzyme and histidine oligopeptide showed optimal activity at pH 6.5 and 25 ℃ and showed the highest stability at pH 6.0. The production of recombinant heparin degrading enzyme was 208,000 U / L in the non-optimized medium, and when immobilized on CNBr-active sepharose 4B, the relative activity was 25-35% when compared with the heparin-degrading enzyme before immobilization . The heparin degrading enzyme comprising histidine oligopeptide and heparin are reacted in a solution state, and after the reaction, the heparin degrading enzyme including histidine oligopeptide is easily removed by the chelating support, and the recombinant heparin degrading enzyme including histidine oligopeptide is chelated When immobilized on a supporter, the activity of 60-80% when compared with the activity before immobilization, the time required for immobilization is 10-30 minutes, and heparin is not needed at all. Thus, the heparin degrading enzyme constituting the histidine oligopeptide is chelated The immobilized enzyme immobilized on a supporter was superior to the conventional method.

Description

Method for producing low molecular weight heparin using recombinant heparin-degrading enzyme comprising histidine oligopeptide

FIG. 1 is a diagram showing a vector having a heparinase gene including a histidine oligopeptide of the present invention, a vector having a heparinase gene, and a process for producing the same.

FIG. 2 is a photograph showing SDS-PAGE (polyacrylamide gel electrophoresis) analysis of heparin-degrading enzyme (lane 2) and heparin-degrading enzyme (lane 1) including the histidine oligopeptide prepared according to the present invention.

FIG. 3A is a graph showing the optimum pH of the heparin-degrading enzyme including the histidine oligopeptide. FIG. 3B is a graph showing the optimal temperature of the heparin-degrading enzyme including the histidine oligopeptide.

3C is a graph showing the pH stability of the heparin-degrading enzyme including the histidine oligopeptide.

FIG. 4 is a graph showing the results of comparing the activities of heparinase (.circle-solid.) And heparinase (.circle-solid.) Immobilized on CNBr-active sepharose 4B.

FIG. 5 is a graph showing the results of comparing the activities of heparinase (.circle-solid.) Containing a histidine oligopeptide immobilized on a chelating support and heparinase (.smallcircle.) Containing a histidine oligopeptide.

Industrial use

The present invention relates to a method for producing a low molecular weight heparin using a recombinant heparin degrading enzyme comprising a histidine oligopeptide, and more particularly, to a combined vector comprising a gene of a recombinant heparin degrading enzyme comprising a histidine oligopeptide, The present invention relates to a method for producing a low molecular weight heparin by enzymatic degradation of heparin using a heparin degrading enzyme comprising a recombinant heparin degrading enzyme comprising a histidine oligopeptide produced by using the same and a produced histidine oligopeptide.

Conventional technology

Heparin is a glucosaminoglycan composed of a linear polysaccharide formed from D-glucosamine and hexuronic acid with α1 → 4 bonds. Its molecular weight is 5,000-30,000 and its size varies. Its average molecular weight is 12,000-15,000. Heparin binds to antithrombin III and causes structural changes. It acts on thrombin, FXa, FIXa, FXIa, and calicaine, which are involved in blood clotting mechanism, to cause anticoagulant action. Among these enzymes, thrombin is most sensitive (J. Hirsh et al., Blood, 79, 1-77 (1992)). These heparin biological effects have been used to prevent post-thrombolytic embolization, but have been accompanied by side effects such as hemorrhage.

Low-molecular-weight heparin is a segment of heparin with an average molecular weight of 4,000 - 6,500 and many differences compared to heparin. The difference in anticoagulation mechanism is due to the fact that the ratio of the activity of anti-IIa to the activity of anti-Xa is almost the same for heparin, while the low molecular weight heparin is an anti- Xa activity is as high as 1: 2-1: 4.

In addition, the difference in the molecular weight of heparin acts on plasma proteins and endothelial cells to neutralize them. Low-molecular-weight heparin acts on plasma proteins, platelets, and endothelial cells at a lower rate than heparin, Thereby increasing bioavailability. In particular, the interaction of heparin with vWF (von Willebrand factor) inhibits vWF-dependent platelet function and causes hemorrhage, which is a side effect of heparin. However, low molecular weight heparin has a low affinity for vWF and thus can reduce the bleeding phenomenon.

Due to the biological utility of these low molecular weight heparins, methods have been devised for the production of low molecular weight heparin from heparin in a variety of ways. The method has been made by methods such as nitrite depolymerization, benzylation and alkaline depolymerization, peroxide depolymerization, and enzymatic depolymerization.

Dual enzymatic depolymerization is a method using heparinase. The heparin-degrading enzyme is an enzyme produced from Flavobacterium heparinum which acts on the glycosidic bond between the N-sulfate D-glucosamine of heparin and the sulfate D-glucuronic acid (or L-iduronic acid) α1-4-cleaving enzyme, first isolated by Linker and Hovingh, and then cloned and expressed in E. coli in 1993 (R. sassekharah et al., Proc. Natl Acad Sci USA, 90, 3600 -3664 (1993)). This heparin degrading enzyme is used as an enzyme for converting heparin into a low molecular weight heparin by utilizing the heparin decomposability.

Previously, heparin degradation enzymes and heparin were reacted together to form low molecular weight heparin, and heparin degradation enzymes were immobilized on CNBr-active sepharose 4B to decompose heparin (USP 5,106,734, H. Bernstein et al. , Methods Enzym, 137, 515-529 (1988)). In particular, the method of immobilizing the heparinase is easy to recover the enzyme after the reaction between the enzyme and the heparin and can be reused, and it also affects the stability of the heparinase, which greatly contributes to stabilization of the enzyme.

To date, as a support for immobilizing the heparin-degrading enzyme, CNBr-active sepharose 4B, chlorinated tresyl-active sepharose, carbidimide, or active ester-active CM-sepharose, CM- cellulose, polyacrylamide, epoxy Activated sepharose 4B (H. Bernstein et al., Methods Enzym, 137, 515-529 (1987)) have been attempted, and the highest activity of immobilized heparin degrading enzymes has been demonstrated 1988).

However, the method of immobilizing heparin degrading enzyme in CNBr-active sepharose 4B is not only time consuming for about 40 hours, but also inhibits heparin degradation enzyme in CNBr-active sepharose 4B, (Linhart, RJ, et al., Biochim. Biophys. Acta, 702, 192-203 (1982), VC Yang, et al., J. Biol. Chem., 260, 1849-1857 (1985)), there was a disadvantage in that a large amount of heparin was added to protect the active site with heparin and the activity of immobilized heparinase was reduced.

The disadvantages of such time consuming, heparin addition, and reduction of heparin degrading enzyme activity were solved by immobilizing the heparin degrading enzyme including the histidine oligopeptide on the chelating support. In addition, when the heparin and heparin degrading enzyme are reacted together in a solution state and heparin degrading enzyme is removed when separating the low molecular weight heparin, the difficulty in removing the heparin degrading enzyme is reduced by using heparin degrading enzyme including histidine oligopeptide, The removal of the heparin degrading enzyme containing the peptide was solved by facilitating the separation of the low molecular weight heparin.

[Object of the invention]

It is an object of the present invention to provide a recombinant vector comprising a gene of a recombinant heparin degrading enzyme comprising a histidine oligopeptide and a recombinant heparin degrading enzyme comprising a histidine oligopeptide prepared using the recombinant vector.

In addition, the present invention relates to a heparin-degrading enzyme, which comprises enzymatically decomposing heparin to produce a low molecular weight heparin, and using the heparin degrading enzyme comprising a histidine oligopeptide, the reaction is terminated in a solution state and then heparin degrading enzyme comprising histidine oligopeptide Of heparin-degrading enzyme and immobilized heparin-degrading enzyme when immobilized heparin-degrading enzyme is used, a method of immobilizing heparin-degrading enzyme including histidine oligopeptide, which is superior in terms of time and efficiency, And then fixing the heparin on the supporter to enzymatically decompose the heparin to produce a low molecular weight heparin.

SUMMARY OF THE INVENTION [

The present inventors have conducted studies to remove the heparin-degrading enzyme after the enzymatic reaction of heparin and heparin-degrading enzyme in the above-mentioned solution state and to overcome the disadvantages of the heparin-degrading enzyme immobilization when the heparin and the heparinase are used for the heparin- , A vector having a heparin degrading enzyme gene containing a histidine oligopeptide was prepared, and a heparin degrading enzyme comprising a histidine oligopeptide was prepared using the E. coli transformed with the vector, and a histidine oligopeptide After heparin-degrading enzyme was reacted in heparin solution, heparin degrading enzyme including histidine oligopeptide was easily separated by chelating support and heparin degrading enzyme including histidine oligopeptide was immobilized on chelating support And decomposed by using this enzyme of heparin to prepare a low molecular weight heparin.

The present invention provides a vector (pE16b-hepa2) into which a heparinase gene containing a histidine oligopeptide is inserted between the NcoI and BamHI recognition sites of pET16b.

The present invention also provides an Escherichia coli transformant strain in which the above-mentioned vector is introduced into Escherichia coli.

The Escherichia coli is preferably BL21 (DE3).

BL21 (DE3) transformed with the above pE16b-hepa2 vector was deposited with the KFCC under accession number KFCC-10903 on Jul. 3, 1996.

Also, the present invention provides a method for preparing a heparin-degrading enzyme comprising a histidine oligopeptide using the E. coli transformant.

The present invention also provides a method for immobilizing a heparin-degrading enzyme comprising a histidine oligopeptide.

The present invention also provides a method for preparing low molecular weight heparin by enzymatic degradation of heparin using a heparin degrading enzyme comprising immobilized histidine oligopeptide.

[Example]

Hereinafter, preferred embodiments of the present invention will be described. However, the following examples are only for the better understanding of the present invention and are not intended to limit the scope of the present invention.

1. Materials and Methods

1) Strain and plasmid used

BL21 (DE3) was used for the expression of Escherichia coli strains used in this study. DH5α (F ^- lacZ M15 hsdR17 (r ^- m ^- ) gyrA36) was used as the Escherichia coli strain for general gene manipulation and plasmid isolation.

PUC19 was used as a plasmid for cloning the heparinase gene. As plasmids for expression in E. coli, pET11a and pET16b (Novagen's No. 69436-1, 69662-1) were used.

2) Restriction enzymes and reagents

Restriction enzymes for DNA recombination, T4 DNA ligase, calf-long alkaline phosphatase, and ribonuclease were purchased from NEB. Separation and purification of DNA from agarose gel was performed by purchasing GENE CLEAN II and MERMAID kit from Bio101

3) Method

DNA recombination was performed according to the method of Maniatis et al. (Sambrook, J. et al., Molecular Cloning A Laboratory Manual, 2nd ed. (1989)). Transformation of E. coli was performed according to the method of Inoue et al. , Gene, 96, 23-28 (1990)).

The activity of heparinase was measured using UV 232 nm method and azir A method. UV 232 nm method and a constant temperature water bath fixed at 25 ° C in a JASCO V-550 spectrophotometer were used to fix the temperature and the degree of change in absorbance with time was measured using UV 232 nm. Heparin was prepared in a concentration of 12.5 mg / ml in 50 mM sodium phosphate (pH 7.1), 100 mM NaCl, 1 ml was taken, and a certain amount of enzyme solution was added to investigate the change in absorbance (H. Bernstein et al., Methods Enzym, 137 , 515-529 (1988)). In addition, the method using azur A was performed by reacting heparin and heparinase with each other, adding 5 μl of the reaction solution to 5 ml of 0.02 mg / ml Ajuar A staining solution, suspending and measuring the absorbance at 620 nm to obtain 0-8 mg heparin / Ml < / RTI > was made and compared to determine the degraded amount of heparin. The activity of the heparin-degrading enzyme was 1 unit of the amount of heparinase capable of degrading 1 mg of heparin per hour (P. M. Galliner et al., Appl. Environ Microbiol., 41, 360-365 (1981)).

The bioactivity of anti-FXa was measured by the COATEST low-molecular-weight heparin kit from Chromogenics Inc. using the national low-molecular-weight heparin standard from National Institute of Biological Standards and Controls, London (NIBSC) as a control.

2. Cloning of heparinase gene from F.

Based on the nucleotide sequence of the discovered heparin-degrading enzyme (gene bank database accession No. L12534), oligomers (oligomer 1) complementary to the 5 'nucleotide sequence of heparin degrading enzyme and NdeI restriction enzyme site and 3' An oligomer (oligomer 2) to which a restriction enzyme site of BamHI complementary to the sequence was added was synthesized.

Oligomer 1: 5'-GACATATGAAAAAACAAATTCTA-3 '

Oligomer 2: 5'-AGGATCCTTACTATCTGGCAGTTTC-3 '

F. heparinum was cultured in a nutrient broth at 30 ° C for 48 hours, and the precipitated cells were suspended in distilled water by centrifugation of the stock solution, and 2 μl of the cell suspension and 100 μl of the oligomer (10 μl) The PCR product was obtained as a reaction mixture, and the obtained PCR products were electrophoresed on 0.8% agarose gel, treated with NdeI and BamHI, treated with NdeI and BamHI, and ligated to produce pU-hepa2 ).

3. Preparation of heparinase expression vector pE16b-hepa2 containing the heparinase-degrading enzyme expression vector pE-hepa2 and the histidine oligopeptide The heparinase gene was isolated from the pU-hepa2 thus prepared, and pET11a and pET16b (manufactured by Novagen Co., Ltd .; 69436-1, and 69662-1), pE-hepa2 and pE16b-hepa2, which are recombinant heparin degrading enzyme expression vectors containing recombinant heparin degrading enzyme and histidine oligopeptide, were prepared, respectively Degree). In the case of expression vector pE-hepa2, T7 promoter, heparin degradation under the control of T7 transcription termination factor

The gene of the enzyme is located, and in the case of the expression vector pE16b-hepa2, the gene of the recombinant heparinase containing the histidine oligopeptide is located under the control of the T7 promoter and T7 transcription termination factor, and the histidine oligopeptide (10 histidine residues) Attached to the amino terminus of the enzyme.

4. Expression of heparin-degrading enzyme containing heparin-degrading enzyme and histidine oligopeptide pE-hepa2, a recombinant heparinase-degrading enzyme expression vector, and pE16b-hepa2, a recombinant heparinase-degrading enzyme expression vector containing histidine oligopeptide, And the expression of heparinase was confirmed. Cells having the respective vectors were induced with IPTG (isopropyl-beta-D-thiogalactoside), and then the culture was centrifuged to precipitate the cells, and the cells were precipitated by the Laemmli method (Laemmli, UK Nature, 227, 680-685 As shown in FIG. 2, both strains having pE-hepa2 and pE16b-hepa2 showed heparin-degrading enzyme (lane 1 (SEQ ID NO: ) And heparin degrading enzyme (lane 2) including a histidine oligopeptide. Lane 3 is a size marker.

E. coli BL21 (DE3) transformed with the above pE-hepa2 vector was deposited with the KFCC under accession number KFCC-10903 on Jul. 3, 1996.

5. Activity of heparinase containing recombinant heparin-degrading enzyme and histidine oligopeptide A strain producing heparinase containing heparinase and histidine oligopeptide, respectively, was cultured in the presence of 1000 ml of an unoptimized medium (Luria broth) Ml < / RTI > flask for 3 hours at 30 < RTI ID = 0.0 > C < / RTI > The culture was centrifuged and the precipitated cells were disrupted with an ultrasonic disrupter and centrifuged. The precipitate was washed with Triton-X-100 solution and dissolved in 6M guanidine-HCI to a protein concentration of about 20 mg / ml. And the activity was examined again by re-returning to the re-return buffer. The activity of the recombinant heparinase was about 208,000 U / L and that of the heparinase containing histidine oligopeptide was about 160,000 U / L. These results show that the production was superior to that of 100,000 U / L prepared from F. heparinum under the conditions of the optimum medium of USP 4,443,545.

6. Changes in pH and Activity of Heparin Degrading Enzyme Including Histidine Oligopeptide The activity of the recombinant heparin degrading enzyme including histidine oligopeptide was measured by the inclusion body Lt; RTI ID = 0.0 > recombinant < / RTI > heparinase containing the histidine oligopeptide. The recombinant heparinase containing the histidine oligopeptide was added to the heparin-containing citrate-phosphate buffer solution at pH 4, 5, 6 and 7, the Tris-HCl buffer at pH 8 and 9, respectively, and UV 232 nm And the relative activity thereof is shown in FIG. 3A. As shown in FIG. 3A, it showed the highest activity at pH 6.5 and showed a desirable activity at pH 5 to 8. This result is similar to the results of heparin degrading enzyme isolated from F. heparinum of P. Hovingh et al. (P. Hovingh, et al., J. Biol. Chem. 245, 6170-675 C. Yang et al. (VC Yang, et al., J. Biol. Chem., 260, 1849-1857 (1985)). On the other hand, the activity of the recombinant heparinase was examined at the same pH of citrate-phosphate buffer, Tris buffer, and sodium phosphate buffer. As a result, it showed the highest activity in the sodium phosphate buffer solution.

Claims (6)

A method for producing a low molecular weight heparin by reacting a heparin degrading enzyme comprising a histidine oligopeptide prepared using E. coli transformed with an expression vector comprising a heparin degrading enzyme gene comprising a histidine oligopeptide with heparin. The method according to claim 1, wherein the E. coli is BL21 (DE3) (KFCC-10903) transformed with a pE16b-hepa2 vector. The method of claim 1, wherein the heparin degrading enzyme comprising a histidine oligopeptide is reacted with heparin in a vector immobilized on a chelating support. The method of claim 1, wherein the heparin degrading enzyme comprising histidine oligopeptide is reacted with heparin and then immobilized on a chelating support to separate low molecular weight heparin. The method according to claim 3 or 4, wherein the pH is between 5.0 and 8.0 and the temperature is between 20 ° C and 28 ° C. The method for producing low molecular weight heparin according to claim 3 or 4, wherein the heparin degrading enzyme comprising a histidine oligopeptide is immobilized on a chelating support using any one of zinc, copper, and nickel ions. ※ Note: It is disclosed by the contents of the first application.