Method for preparing recombinant heparinase III by utilizing SUMO fusion expression system and SUMO_heparinase III fusion protein prepared by method

A fusion protein and fusion expression technology, applied in the biological field, can solve the problems of reducing the yield of the target protein and interfering with the normal function of the target protein, and achieve the effects of improving stability and solubility, promoting correct folding, and improving purification efficiency

Pending Publication Date: 2021-06-25
上海宝维医药技术有限公司
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Problems solved by technology

However, fusion protein tags such as glutathione sulfhydryltransferase tag (GST-Tag), maltose-binding protein tag (MBP-Tag), transcriptional anti-termination factor A tag (NusA-Tag) and other fusion protein tags, because of their large molecular weight (26- 55kDa), which will interfere with the normal function of the target protein, and the removal of tags during the expression and subsequent purification process will relatively reduce the yield of the target protein

Method used

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  • Method for preparing recombinant heparinase III by utilizing SUMO fusion expression system and SUMO_heparinase III fusion protein prepared by method
  • Method for preparing recombinant heparinase III by utilizing SUMO fusion expression system and SUMO_heparinase III fusion protein prepared by method
  • Method for preparing recombinant heparinase III by utilizing SUMO fusion expression system and SUMO_heparinase III fusion protein prepared by method

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Embodiment 1

[0055] 1. Plasmid transformation into BL21(DE3) competent cells

[0056] 1.1 Add 2 μL of the plasmid to 100 μL of competent bacteria and place on ice for 30 minutes;

[0057] 1.2 Heat shock at 42°C for 90s, quickly place on ice for 5min; add 500μL LB culture solution; 1.3 Shake at 37°C, 220rpm for 1h, centrifuge and spread all on the resistant LB plate, and incubate overnight at 37°C.

[0058] 2. Expression Identification

[0059] 2.1 Pick 5 single clones on the plate and inoculate them in a test tube containing 4mL LB culture medium with an appropriate amount of resistance;

[0060] 2.2 Shake at 220 rpm at 37°C until the OD600 of the bacteria is 0.6-0.8;

[0061] 2.3 Take out 1 mL of the culture, centrifuge at 12000 g for 5 min at room temperature, discard the supernatant, resuspend the bacterial pellet with 80 μL of 1×PBS buffer and add 20 μL of 5×Loading Buffer;

[0062] 2.4 Add IPTG to the remaining culture to a final concentration of 0.5 mM, shake at 220 rpm at 37°C fo...

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Abstract

The invention discloses a method for preparing recombinant heparinase III by utilizing an SUMO fusion expression system and the heparinase III prepared by the method, the preparation method comprises the following steps: selecting a heparinase III sequence from Pedobacter heparinus, wherein the amino acid sequence of the heparinase III is shown as SEQ ID No.1, and the total number is 659aa; removing a signal peptide sequence to obtain a DNA sequence of the heparinase III, such as SEQ ID No.2; inserting the DNA sequence of the heparinase III into a pSMART vector plasmid with an N-terminal SUMO protein tag; transforming the correct plasmids into BL21 (DE3) escherichia coli competent cells, selecting monoclone, and obtaining fusion protein through fermentation and purification; and cutting the SUMO tag protein from the fusion protein by using the SUMO protease to obtain the heparinase III. The method has the advantages that the solubility of the target protein is improved, correct folding of the target protein is promoted, and inclusion bodies are prevented from being formed; the purification cost is reduced and the purification efficiency is improved; the molecular weight of the SUMO protein tag is small, the influence on the enzymatic activity of the heparinase III is small, and the purified heparinase III is obtained.

Description

[0001] The invention relates to the field of biotechnology, in particular to a method for preparing recombinant heparinase III (heparinase III) using a SUMO fusion expression system. Background technique [0002] Heparinase is mainly isolated from some bacteria that use heparin as a carbon source, initially from Pedobacter heparinus. There are three kinds of heparinase isolated and purified from Flavobacterium heparinus, namely heparinase I, Heparanase II, Heparanase III. The main function of heparinase is to degrade heparin, and it is widely used in the production of low-molecular-weight heparin and ultra-low-molecular-weight heparin by enzymatic degradation. [0003] At present, there are two main sources of heparinase products used in the production of low molecular weight heparin: one is directly fermented and extracted from microorganisms producing heparinase (Shenzhen Haipurui Pharmaceutical Co., Ltd., CN102286448B); the other is through genetic engineering technology C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70
CPCC07K2319/95C12N9/2402C12N15/70C12Y302/01019
Inventor 蒋建华张亮邢岭吴双高伟
Owner 上海宝维医药技术有限公司
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