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A kind of mbp fusion heparanase Ⅱ and its coding gene and preparation method

A technology of heparanase and coding sequence, which is applied in the field of MBP fusion heparanase II and its coding gene and preparation, can solve the obvious effect of catalytic performance, and the catalytic ability and thermal stability of heparanase II enzyme have not been significantly improved and other problems, to achieve the effect of improving catalytic activity and thermal stability

Active Publication Date: 2020-03-17
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

In 2009, Lien-I Hor et al. (Lien-I Hor, Howard A. Shuman, Genetic analysis of periplasmic binding protein dependent transport in Escherichia coli. J MolBiol, 1993,233:659-670) analyzed the different sources of heparanase Ⅱ Through the analysis of the primary sequence and advanced structure, it was found that changing the charge properties of several conservative amino acid residues Glu207 and His411 in heparanase Ⅱ can effectively improve the affinity of heparanase Ⅱ to heparan sulfate, but it has no effect on Catalytic properties have a noticeable effect
It can be seen that the catalytic ability and thermal stability of heparanase II have not been significantly improved at present, and there is no effective method to greatly improve the catalytic activity and thermal stability of heparanase II through genetic engineering.

Method used

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  • A kind of mbp fusion heparanase Ⅱ and its coding gene and preparation method
  • A kind of mbp fusion heparanase Ⅱ and its coding gene and preparation method
  • A kind of mbp fusion heparanase Ⅱ and its coding gene and preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0039]Example 1 Optimization of rare codons of heparinase II and construction and expression of MBP fusion heparinase II vector

[0040] 1. Heparinase II rare codon optimization

[0041] The coding region sequence of heparinase II from Flavobacterium heparin contains a large number of rare codons, which will seriously affect the protein translation efficiency when expressed in Escherichia coli. See figure 1 .

[0042] According to the codon usage preference of Escherichia coli, the coding region sequence of heparinase II was optimized with the current mainstream codon optimization software DNA2.0 (downloaded from https: / / www.dna20.com / official website). The protein sequence of heparinase II remains unchanged, and only the degeneracy of codons is used. Codons that are used less frequently in Escherichia coli and will affect the efficiency of ribosome passage during translation will be used with higher frequency. The codons were replaced to obtain the optimized heparinase II...

Embodiment 2

[0049] Example 2 Site-directed mutation and analysis of amino acid at 1126 site of MBP fusion heparinase II

[0050] 1. MBP fusion heparinase II 1126 site modification basis

[0051] The coding region sequences of heparinase II from two strains of Flavobacterium heparin were analyzed, and there was only one amino acid difference between the two protein amino acid sequences at position 758: P758A. According to the above rare codon optimization method and the construction method of MBP fusion heparinase II vector, the MBP fusion heparinase II expression vector with only one amino acid site difference (P1126A) was constructed. The above two vectors were transformed and expressed to obtain MBP-HepB fusion proteins HepB (1126P) and HepB (1126A).

[0052] Protein extraction and purification process: 100ml of the target Escherichia coli fermentation bacteria liquid was collected and resuspended with 20ml of protein extraction buffer, ultrasonically disrupted the bacteria, the crushe...

Embodiment 3

[0070]Example 3 MBP-fused heparinase II has a similar action site to the heparinase II 1126 site in the MBP-fused heparinase II vector in Example 2

[0071] At present, there is no structural information of MBP-fused heparinase II in the PDB database, only the structural information of heparinase II. The 758 site of heparinase II was analyzed by Discover Studio 2.5, and this site is now on the binding surface of the heparinase II protein dimer (see Figure 7 ). Depend on Figure 7 and Figure 8 Comparative analysis showed that there was basically no difference in spatial conformation after amino acid substitution at position 758.

[0072] Using this method, the interaction force of the amino acids constituting the dimer binding surface was analyzed, and the results showed that among the 33 amino acid residues found to constitute the dimer binding surface, there were strong interactions between some amino acid residues. effect, some with slightly weaker interactions (see ...

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Abstract

The invention discloses an MBP fusion heparinase II. The MBP fusion heparinase II is a protein a) composed of an amino acid sequence represented by sequence 1 in a sequence b, or a protein b) obtained after site-specific mutagenesis of one or more of 33 amino acid sites forming the heparinase II dimer binding surface in the MBP fusion heparinase II protein with the amino acid sequence represented by the sequence 1 in the sequence table. The invention also discloses an encoding gene and a preparation method of the protein. The MBP fusion heparinase II with obviously improved catalysis activity and thermal stability is finally obtained through site-specific mutagenesis of the MBP fusion heparinase II through gene engineering means.

Description

technical field [0001] The invention relates to the fields of genetic engineering and fermentation engineering, in particular to an MBP fusion heparinase II and its encoding gene and a preparation method. Background technique [0002] Heparinase is a type of polysaccharide lyase that acts on heparin or heparansulfate. Heparinase II (No EC code) and heparinase III (EC4.2.2.8). [0003] Compared with heparinase I and III, heparinase II has a wider range of substrates and action sites, and the types and compositions of products are also more diverse. This feature makes it have higher polysaccharide degradation efficiency. It plays an important role in the preparation of molecular weight heparin and the precise structural analysis of heparin. [0004] Due to the important application value of heparinase II, researchers have been working on improving the catalytic performance of heparinase II through molecular modification. In 2009, Lien-I Hor et al. (Lien-I Hor, Howard A. Shu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/66C12N15/09
Inventor 邢新会苏楠张翀
Owner WUXI RES INST OF APPLIED TECH TSINGHUA UNIV
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