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L-asparaginase variant with increased activity

An asparaginase and variant technology, applied in the field of medical bioengineering, can solve the problems of aggravating the patient's economic burden, pyrogen reaction, high price and the like

Active Publication Date: 2013-04-24
BEIJING ABZYMO BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the L-ASP enzyme activity clinically used is low, and the half-life in the body is short (<5 hours), so the dosage is very large (30-50 mg), and frequent injections (3 times a week) are required, which often cause allergic reactions and pyrogens in patients. Reactions and other side effects [4]
Although PEGylated L-ASP has a significantly prolonged half-life, the overall incidence of allergic reactions is lower than that of natural extracts, but PEGylation causes a 30% reduction in enzyme activity, and 11% of patients who are not allergic to natural L-ASP are sensitive to PEG Hypersensitivity to chemical products (data source is the product manual of PEGylated L-ASP Oncaspar from Enzon Company)
In addition, PEGylated L-ASP is expensive, which increases the economic burden of patients, so the current treatment still mainly uses natural products extracted from Escherichia coli
The above-mentioned deficiencies make the application of L-ASP limited

Method used

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  • L-asparaginase variant with increased activity
  • L-asparaginase variant with increased activity
  • L-asparaginase variant with increased activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: Cloning of Escherichia coli L-ansB gene

[0045] According to the L-ansB nucleotide sequence published by NCBI, the upstream primer mansB-001 (5'-CG GAATTC ATGGAGTTTTTCAAAAAGACG-3') (SEQ ID NO: 1) and downstream primer mansB-002 (5'-CG GGATCC TTAGTACTGATTGAAGATCTG-3') (SEQ ID NO: 2), where the underlines show the additionally added EcoRI and BamHI restriction sites, and two additional protective bases are added at the 5' end of the primer.

[0046] PCR reaction was carried out with E. coli DH5α genome as template. The reaction conditions were: pre-denaturation at 95°C for 5 min, followed by 30 cycles of reaction according to the following parameters: denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 90 s, and finally extension at 72°C for 10 min.

[0047] After the PCR product was subjected to 1% low-melting point agarose gel electrophoresis, the gel piece containing the target fragment was cut out and placed in a 1.5mL E...

Embodiment 2

[0053] Embodiment 2: Construction of recombinant expression construct pBV-ansB

[0054] Carry out EcoRI and BamHI (Bao Biological Engineering (Dalian) Co., Ltd.) double enzyme digestion reaction on the sequencing construct pMD18T-ansB in Example 1 to recover the L-ansB target gene fragment:

[0055]

[0056] After mixing, the enzyme digestion reaction solution was placed at 37° C. for overnight incubation, and then purified and recovered by the method in Example 1. The pBV220 expression vector was digested with EcoRI and BamHI in the same manner, and the linear pBV220 expression vector fragment was purified and recovered.

[0057] The purified L-ansB target gene fragment and the linear pBV220 expression vector fragment were ligated according to the following system:

[0058]

[0059]

[0060] The ligation reaction solution after mixing was placed at 25°C for 30 minutes, and then used at 4°C for later use or directly for transformation reaction.

[0061] Add 10 μL of...

Embodiment 3

[0062] Example 3: Error-prone PCR random mutation of L-ansB gene

[0063] Prepare the error-prone PCR reaction solution according to the following system:

[0064] 10 μL of 10×PCR buffer

[0065] ·50×dNTPs mixture 2μL

[0066] · 10mM dCTP 2μL

[0067] · 10mM dTTP 2μL

[0068] 10×MgCl 2 10μL

[0069] · MnCl 2 5μL

[0070] ·Upstream primer mansB-001 2μL

[0071] ·Downstream primer mansB-002 2μL

[0072] ·Template pBV-ansB 10~100ng

[0073] · rTaq DNA polymerase 1μL (5U)

[0074] Add double distilled water to 100μL

[0075] in:

[0076] 10×PCR buffer: 20mM MgCl2, 200mM KCl, 50mM Tris-HCl, pH8.3;

[0077] 50×dNTPs mixture: containing 10mM each of dATP, dGTP, dTTP, and dCTP;

[0078] 10×MgCl 2 Solution: 70mM, dissolved in sterile water;

[0079] MnCl 2 Solution: 1mM, dissolved in sterile water;

[0080] rTaq DNA polymerase: purchased from Treasure Bioengineering (Dalian) Co., Ltd., catalog number DR001.

[0081] The error-prone PCR rea...

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Abstract

The invention relates to a L-asparaginase variant with increased activity, and is a variant of escherichia coli wild type L-asparaginase shown in a SEQ ID NO:3, and the variant comprises an one or a plurality of amino acid substituted amino acid sequence on 48th site, 49th site, 152nd and 283rd by corresponding to a SEQ ID NO:3. The invention also provides the separated nucleic acid containing the nucleotide sequence which codes the L-asparaginase variant, a recombinant expression construct containing the nucleic acid and a recombinant host cell containing the expression construct. In addition, the invention also provides a method for generating the L-asparaginase variant. The invention also provides a pharmaceutical composition used for treating tumor containing the L-asparaginase variant.

Description

technical field [0001] The invention belongs to the technical field of medical bioengineering. In particular it relates to L-asparaginase variants with increased activity and the use of these variants in the treatment of tumors. Background technique [0002] Leukemia is a malignant disease that seriously threatens human life in the world today, accounting for the sixth in the incidence of tumors and the first in the incidence of malignant tumors in adolescents [1]. Among them, acute lymphocytic leukemia (ALL) is a hematological malignancy with a high incidence rate, especially in adolescents and children, and its incidence rate accounts for about 25% of all adolescent cancer incidences. rate of about 80% [2]. Without treatment, patients die within months or even weeks due to the dramatic increase and spread of malignant cells. [0003] Among various treatment methods, L-asparaginase (L-ASP, EC 3.5.1.1) is an important chemotherapy drug for leukemia and an important part o...

Claims

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Application Information

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IPC IPC(8): C12N9/82C12N15/55C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K38/50A61P35/00A61P35/02
CPCA61K38/50A61P35/00A61P35/02C12N9/82C12N15/63C12Y305/01001
Inventor 沈林李鼎锋孙志丹陈星梅刘勇
Owner BEIJING ABZYMO BIOSCIENCES CO LTD
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