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A connecting peptide and its application

A technology for linking peptides and heparinase, applied in the fields of genetic engineering and fermentation engineering

Active Publication Date: 2019-04-09
WUXI RES INST OF APPLIED TECH TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the connecting peptide has an important influence on the function of the fusion protein, it is regrettable that so far, there are few studies on the connecting peptide in polysaccharide lyases such as heparanase and chondroitin sulfate, and the existing related enzymes are far from the requirements of industrial applications. larger distance

Method used

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  • A connecting peptide and its application
  • A connecting peptide and its application
  • A connecting peptide and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Design of linker peptide, construction and expression of target linker peptide MBP fusion polysaccharide lyase vector

[0048] 1. Design of connecting peptide

[0049] (1) The origin of the connecting peptide:

[0050] Since the length and properties (rigidity and flexibility) of the connecting peptide can significantly affect the properties of the fusion protein, this is mainly due to the change of the spatial distance and positional relationship between the two elements in the fusion protein, resulting in the mutual force and space The difference in steric hindrance will affect the structure of the protein to different degrees, and finally affect the biological function of the two elements, especially the biological function of the main catalytic enzyme. Length has the most significant impact on the properties of fusion proteins. However, for this reason, it is difficult to predict to what extent changes in length will affect fusion proteins, so it is very ...

Embodiment 2

[0055] Expression, purification and enzyme activity analysis of embodiment 2 target protein

[0056] Expression of polysaccharide lyase vector containing target connecting peptide fusion: transform the constructed plasmid containing target connecting peptide fusion polysaccharide lyase vector into Escherichia coli respectively, and shake the strain transformed with recombinant plasmid in LB medium containing 100ug / mL ampicillin After culturing overnight (37°C, 180rpm, 14-16h), inoculate into LB / M9YE fermentation medium at a ratio of 1:100, shake at 37°C and 180rpm to the logarithmic growth phase, add IPTG and turn to 200rpm, 15°C Low temperature induction culture for 20-22h.

[0057] Protein extraction and purification process: Collect 100ml of the target Escherichia coli fermentation broth and resuspend with 20ml of protein extraction buffer, ultrasonically disrupt the bacteria, centrifuge the broken product at 10,000rpm for 30 minutes at 4°C, collect the supernatant, and fil...

Embodiment 4

[0093] Example 4 Thermal stability analysis of MBP fusion polysaccharide lyase before and after the replacement of the connecting peptide

[0094] Measure the enzyme activity immediately after protein purification as the enzyme activity at time 0, place the pure enzyme in a water bath at the detection temperature, select different time points to sample and measure the enzyme activity, compare with the enzyme activity at time 0, and calculate the time Relative to the percentage of enzyme activity at time 0. Continue this operation until the enzyme activity drops to a lower level, so that a series of corresponding relationship points between warming time points and remaining enzyme activities are obtained, and these points are connected to obtain the inactivation curve of the enzyme at this temperature, that is, The stability of the enzyme at this temperature can be evaluated.

[0095] 1. Analysis of enzyme thermostability after shaking flask fermentation before and after repla...

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Abstract

The invention discloses a connecting peptide and an application thereof. The connecting peptide is mainly applied in preparation of MBP fusion polysaccharide lyases, and in particular, relates to MBP fusion heparinases I, II and III and MBP fusion chondroitinases B and AC. In an MBP fusion expression system, MBP solubilization assisting and folding assisting are improved through application of the connecting peptide, the target protein activity and thermal stability are increased, and the MBP fusion heparinases I, II and III and the MBP fusion chondroitinases B and AC having fermentation enzyme activity in escherichia coli preliminarily reaching an industrial application level and having excellent thermal stability are eventually obtained.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and fermentation engineering, and specifically relates to a connecting peptide and its application. Background technique [0002] Heparinase is a class of polysaccharide lyases that can act on the glycosidic bonds of heparin or heparinsulfate molecules. The currently known types of heparanase are Ⅰ, Ⅱ, and Ⅲ, which were discovered from the gram-negative corynebacterium Flavobacterium heparinus. The substrate specificity and mode of action of the three enzymes are different. The substrate of heparanase I is heparin, the substrate of heparanase II is heparin and heparan sulfate, and the substrate of heparanase III is heparan sulfate. Heparanase has important application value in scientific research and application fields. [0003] Chondroitin sulfate enzyme (chondroitinase or chondroitin sulfaeyase, referred to as ChSase) is a kind of chondroitin sulfate, chondroitin, hyaluronic acid and other ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12N15/11C12N9/88
Inventor 邢新会苏楠张翀
Owner WUXI RES INST OF APPLIED TECH TSINGHUA UNIV
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