Fusion protein, as well as efficient expression method and application thereof

A fusion protein and high-efficiency expression technology, applied in the field of fusion proteins, can solve problems such as difficult to achieve high-efficiency expression

Pending Publication Date: 2020-07-07
BEIJING UNIV OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Both AtSUS1 and RsUGT72B14 genes are derived from plants, and the codon usage preferences of the host Escherichia coli are quite different, so it is difficult to achieve high-efficiency expression by conventional methods

Method used

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  • Fusion protein, as well as efficient expression method and application thereof
  • Fusion protein, as well as efficient expression method and application thereof
  • Fusion protein, as well as efficient expression method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] The construction, expression, in vitro enzymatic catalysis and detection of fusion protein gene recombinant Escherichia coli, the specific implementation steps are:

[0030] (1) AtSUS1 and RsUGT72B14 gene optimization: On the premise of not changing the amino acid sequence, the E. coli preferred codon optimized sequence was used.

[0031] The optimized AtSUS1 nucleotide sequence is shown in SEQ ID NO.2;

[0032] The optimized nucleotide sequence of RsUGT72B14 is shown in SEQ ID NO.3, and the GenBank number is KU523897.1.

[0033] (2) Construction of AtSUS1-RsUGT72B14 fusion gene: Between AtSUS1 and RsUGT72B14 optimized gene sequences, a linker peptide sequence GGTGGATCCGGT including restriction site BamH I (GGATCC) and encoding four amino acids of GGSG was designed; SEQ ID NO.4. The AtSUS1-RsUGT72B14 fusion protein gene was synthesized according to the sequence of SEQ ID NO.1.

[0034] The AtSUS1 gene, RsUGT72B14 gene and AtSUS1-RsUGT72B14 fusion protein gene synthesi...

Embodiment 2

[0043] The construction, expression, and whole-cell catalysis and detection of fusion protein gene recombinant Escherichia coli, the specific implementation steps are:

[0044] Perform the same steps (1)-(3) in Example 1 to obtain recombinant Escherichia coli, then induce expression for 6 hours, adjust the temperature to 30°C for reaction, add 200 μM tyrosol, 200 μM sucrose, and 10 μM UDP, continue to cultivate for 8 to 12 hours, collect and culture After ultrasonic crushing of the liquid, freeze-drying treatment, the freeze-dried powder is extracted with an equal volume of methanol, and after the impurity is passed through the membrane, it is detected by HPLC with the step (6) of Example 1, and the result ~ 4min peak (such as Figure 4 c), indicating that the AtSUS1-RsUGT72B14 fusion protein can also catalyze the synthesis of salidroside in Escherichia coli cells. At the same time, under the same reaction conditions, the recombinant Escherichia coli containing only the RsUGT7...

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Abstract

The invention discloses fusion protein, as well as an efficient expression method and application thereof and belongs to the technical field of fusion protein. The fusion protein is AtSUS1-RsUGT72B14fusion protein, including core fragments of arabidopsis thaliana sucrosesynthaseAtSUS1 and rhodiola sachalinensis uridine diphosphate glucosyl transferase RsUGT72B14. According to the codon preferenceof escherichia coli, an optimized gene sequence of the AtSUS1-RsUGT72B14 fusion protein is obtained by combining the characteristics of gene sequences of AtSUS1 and RsUGT72B14 and introducing GGSG four amino acid connecting peptide sequences by designing a restriction enzyme cutting site connection method, efficient expression of the AtSUS1-RsUGT72B14 fusion protein is realized, and the fusion protein can be used for catalytically synthesizing salidroside.

Description

technical field [0001] The invention relates to the technical field of fusion proteins, and more specifically relates to a fusion protein and its high-efficiency expression method and application. Background technique [0002] Sucrose synthase (SUS) is one of the key enzymes in sucrose metabolism. It can catalyze the reversible reaction of sucrose and UDP to produce fructose and UDPG. Uridine diphosphateglucosyltransferase (UGT) is a modification reaction that catalyzes the transfer of glycosyl groups, and transfers glycosyl groups from activated donor molecules to acceptor molecules. Glycosylation modification is usually the last step in the biosynthesis of natural compounds, which can improve the water solubility, stability and biological activity of natural products. UGT is an important enzyme in the synthesis pathway of glycosyl plant secondary metabolites. For example, UGT72B14 can catalyze the synthesis of salidroside (and by-product UDP) from tyrosol and UDPG. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/62C12N15/70C12P19/44
CPCC12N9/1062C12N9/1066C12N15/70C12P19/44C12Y204/01013C12Y204/01014C07K2319/00C12N2800/22
Inventor 薛飞燕马兰青杨明峰姜梦嫣黄丽娜彭程
Owner BEIJING UNIV OF AGRI
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