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Recombinant clostridium histolyticum type I collagenase as well as preparation method and application thereof

A technology of Clostridium histolyticum and collagenase, which is applied in the field of bioengineering, can solve the problems of complex renaturation process, high technical difficulty, and high production cost, and achieve the effects of facilitating purification, improving purification efficiency, and avoiding renaturation

Pending Publication Date: 2021-06-25
重庆科润生物医药研发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The renaturation process of inclusion bodies is complex, technically difficult, low in yield, and high in production cost, so it cannot be applied to industrial production at present

Method used

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  • Recombinant clostridium histolyticum type I collagenase as well as preparation method and application thereof
  • Recombinant clostridium histolyticum type I collagenase as well as preparation method and application thereof
  • Recombinant clostridium histolyticum type I collagenase as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Recombinant Clostridium histolytica Type I Collagenase Fusion Protein Engineering Bacteria Construction

[0040] 1. Construction of pET30-a(+)-ColG / BL21(DE3) engineering bacteria

[0041] The mature protein of Clostridium histolyticum type I collagenase contains 1008 amino acids, and the gene sequence of Clostridium histolyticum type I collagenase is shown in SEQ ID NO:2.

[0042] The tag protein is selected from the tandem combination of calmodulin-binding peptide and streptavidin-binding peptide; the amino acid sequence of the fusion tag protein is shown in SEQ ID NO: 3, and its nucleotide sequence is shown in SEQ ID NO: 4.

[0043] The linker sequence was designed as -GSGSGGTAMADIGSDDDDK-, and the C-terminus of the above-mentioned CBP-SBP tagged protein was connected to the N-terminus of the mature protein sequence of Clostridium histolyticum type I collagen through the linker. The amino acid sequence of the obtained fusion protein is shown in SEQ ID NO:9 ...

Embodiment 2

[0049] Embodiment 2 Recombinant Clostridium histolyticum Type I Collagenase Engineering Bacteria Fermentation

[0050] Seed liquid activation: divide the liquid LB medium into 500ml shake flasks, 100ml per bottle, kan+160ul, a total of 1 bottle, then absorb 40ul of glycerol seeds, inoculate in LB liquid medium on a shaker overnight at 27°C and 180rpm to cultivate.

[0051] Fermentation process: use 5L Baoxing fermenter for fermentation, set the temperature at 37°C, ventilate at 1.5vvm, add 4ml of trace elements, kan+0.06g sterile filter, then put into the fermenter pH7.0; take LB culture medium stored at 4°C The seeds prepared in the following steps are inoculated into 2L of fermentation medium (M9 improved medium) according to the proportion of 5% (100ml); the initial speed is 200rpm, and the dissolved oxygen speed is set to be linked, and the dissolved oxygen is not less than 30% (the equipment has 3-5% error, need to set the parameter 35%) to correct dissolved oxygen 100%;...

Embodiment 3

[0052] Example 3 Separation and Purification of Recombinant Clostridium histolyticum Type I Collagenase

[0053] 1. Bacterial crushing and clarification

[0054] The bacteria were collected by high-speed freezing and centrifugation, and the bacteria were suspended according to the ratio of 1:10 (1g:10ml buffer: 25mM Tris-HClpH8.0). Break the bacteria 3 times, then refrigerate and centrifuge at high speed, collect the supernatant, and discard the precipitate; use a 0.65 μm hollow fiber column to clarify the bacteriostasis solution by ultrafiltration.

[0055] 2. CBP affinity chromatography

[0056] Take the clarified supernatant and dilute it 2 times with low concentration calcium buffer. The protein of interest was captured with CalmodulinSepharose 4B affinity filler. Elution was performed with 2 mM EGTA (ethylene glycol diethyl ether diamine tetraacetic acid), pH 7.0.

[0057] 3. Enterokinase enzymolysis

[0058] In the above fusion protein solution, 0.5 U of recombinant...

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Abstract

The invention discloses a fusion protein of recombinant clostridium histolyticum type I collagenase. The fusion protein comprises the clostridium histolyticum type I collagenase and tag protein connected to the N end of the clostridium histolyticum type I collagenase. The tag protein comprises a calmodulin binding peptide (CBP) and a streptococcus avidin binding peptide (SBP) which are connected in series. The invention further provides a preparation method and application of the fusion protein of the recombinant clostridium histolyticum I type collagenase. The CBP-SBP is used as a fusion tag, soluble expression of clostridium histolyticum I type collagenase (ColG) can be efficiently promoted, inclusion body renaturation is avoided, purification is facilitated, the production cost is reduced, and the requirements of industrial production can be met.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a high-purity recombinant Clostridium histolyticum type I collagenase and its preparation method and application. Background technique [0002] Collagenase Clostridium Histolyticum (Collagenase Clostridium Histolyticum) is a drug originally developed by BioSpecifics Technologies Group, which was approved by the US Food and Drug Administration (FDA) on February 2, 2010, and obtained by the European Medicines Agency on February 28, 2011 (EMA) approval, and on July 3, 2015, it was approved for marketing by the Japanese Medicines and Medical Devices Agency (PMDA). product name The products of Auxilium Pharmaceuticals (now Endo Pharmaceuticals, Endo International) are sold in the US market, and by Asahi Kasei (Aasahi Kasei) in the Japanese market. product name The products are sold in the European market by Swedish Orphan Biovitrum (SOBI). In addition, the trade name and produc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/52C12N15/62C12N15/70C12N1/21A61K38/48A61P43/00A61P15/00C12R1/19
CPCC12N9/52C12N15/70A61P43/00A61P15/00C07K2319/35A61K38/00Y02A50/30
Inventor 李树刚张伟但国平邓永康李晓丽曹莉君刘涛辛渝杨丹丹于廷和
Owner 重庆科润生物医药研发有限公司
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