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Purified high-specific-activity recombinant batroxobin

A batroxobin, specific activity technology, applied in the field of high specific activity recombinant batroxobin, can solve the problems of high disulfide bond pairing error rate, low specific activity, poor reproducibility, etc.

Active Publication Date: 2008-10-01
SHANGHAI TENRY PHARMCEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is because the error rate of disulfide bond pairing is very high, and almost all inclusion bodies are obtained in prokaryotic cells. Although Japan’s Fujisawa Pharmaceutical Company reported that an active target protein can be obtained through denaturation-refolding of inclusion bodies, its ratio The activity is low and the reproducibility is poor. Up to now, no recombinant batroxobin product has been released by this company

Method used

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  • Purified high-specific-activity recombinant batroxobin
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  • Purified high-specific-activity recombinant batroxobin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Preparation Example

[0088] One, obtain the fermented liquid containing batroxobin protein

[0089] Fermentation of rBAT engineering bacteria in 30L tank:

[0090] Insert the inoculation ratio of 1:10 into a 30L fermenter that has been pre-sterilized and equipped with 15L batch fermentation medium, and carry out fed-batch culture (use ammonia water to control the pH at 4.0, and the temperature is controlled at 30°C). After the carbon source is exhausted (DO rises sharply, within 1 minute), glycerol is added in a stream (the flow rate maintains dissolved oxygen greater than 20%). When the wet weight of the bacteria is about 200 g / L, stop feeding, and after the glycerin is exhausted, add methanol to induce expression (control the pH at 5.8 with ammonia water, and control the temperature at 20°C), by adjusting the rotation speed, tank pressure, air flow and The feeding rate was such that the dissolved oxygen was greater than 20%, and the induction time was 60 hours. At...

Embodiment 2

[0097] performance example

[0098] Detect the rBAT stock solution I prepared in Example 1:

[0099] 1. The main parameters of the experiment:

[0100] Detection instrument model: LCQ DECA XP plus Sampling method: Microspray

[0101] Capillary temperature: 170°C Column: 0.15MM*150MM (RP-C18)

[0102] Instrument company: FINNIGAN Detection method: Positive ion

[0103] 2. Experimental method:

[0104] Sample rBAT was desalted by ultrafiltration, modified with iodoacetamide (IAA) → enzymatic hydrolysis with trypsin and chymotrypsin → enzymatic hydrolysis with N-glycosidase F and F1 → 1 / 2 enzymatic hydrolysis product dithiothreo Sugar alcohol (DTT) reduction, IAA modification→mass spectrometry analysis→data analysis, determine the C-terminal sequence, glycosylation site and disulfide bond pairing mode of rBAT protein.

[0105] 3. Experimental results and analysis:

[0106] 3.1 C-terminal sequence analysis

[0107] The principle of protein C-terminal sequence mass spectrome...

Embodiment 3

[0123] Purification process research example

[0124] 1. Study on hydrophobic chromatography conditions

[0125] Since the fermentation uses an inorganic salt medium (pH value around 6.0), the fermentation supernatant contains higher salt and higher conductivity, so it is desired to use hydrophobic chromatography as the first step of purification to obtain crude and pure rBAT.

[0126] 1.1 Study on Hydrophobic Chromatography Conditions at pH6.0

[0127] Take 100ml of the fermentation supernatant respectively, and proceed as follows:

[0128] Fermentation supernatant volume

(ml)

3M ammonium sulfate volume

(ml)

Volume of water for injection

(ml)

total capacity

(ml)

Ammonium sulfate final concentration

(M)

Group A

100

150

50

300

1.5

Group B

100

100

100

300

1.0

Group C

100

50

150

300

0.5

[0129] For group...

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PUM

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Abstract

The present invention provides a recombined-batroxobin, having following characteristics: (a) the molecular weight is 29-32Kda; (b) at least 90% batroxobin in the recombined-batroxobin has correct 6 pairs of disulfide bonding; Cys7-Cys139, Cys26-Cys42, Cys74-Cys230, Cys118-Cys184, Cys150-Cys163 and Cys174-Cys199; (c) the 146 position and the 225 position in the SEQ ID NO:1 are suffered from glycosylation; (d) the specific activity of the batroxobin is more than 1500KU / mg.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to purified recombinant batroxobin with high specific activity and application thereof. Background technique [0002] As early as 1936, Austrian scholar Van Klobusitzky et al. purified and refined an enzymatic hemostatic agent, Batroxobin, from the venom of the Brazilian spearhead snake (Bothrops atrox). The earliest Chinese translation in China was called "Batroxobin". Batroxobin belongs to the class of serine protease molecules, and its physiological function and molecular size are similar to thrombin (Thrombin), so it is also called "hemocoagulase" later. But with the in-depth research on it, batroxobin has been endowed with different meanings in different periods at home and abroad. There are 5 subspecies of Brazilian spearhead snake (Bothrops atrox), batroxobin extracted from some subspecies has hemostatic effect[1], while batroxobin extracted from other subspecies has the e...

Claims

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Application Information

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IPC IPC(8): C12N9/50A61K38/48A61P7/04C12N9/74
Inventor 黄秀东陈佩新潘学工王强曹之舫
Owner SHANGHAI TENRY PHARMCEUTICAL CO LTD
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