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Method for preparing serratia nuclease through efficient secretory fusion expression and recombination in methylotrophic yeast

A fusion expression, methanol yeast technology, applied in the field of bioengineering, can solve the problems such as the inability to effectively block the biological activity of the target protein, the lack of an extracellular secretion system in Escherichia coli, the high toxicity of host cells, etc. high-efficiency expression

Inactive Publication Date: 2020-06-23
山东仁瑞生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the original intention of the above-mentioned expression system is to utilize the characteristics of good solubility and high expression efficiency of the fusion partner protein to achieve high-efficiency soluble expression of the target protein. The fusion protein can promote the construction and expression of the active target protein, but it cannot effectively block the biological activity of the target protein.
At the same time, because Escherichia coli lacks an extracellular secretion system, the recombinant protein cannot be secreted outside the cell, and most of it accumulates in the cytoplasm
For highly toxic proteins, the higher the expression level of the active form, the more toxic it is to the host cell, and it cannot be used for high-efficiency expression preparation of toxic large proteins

Method used

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  • Method for preparing serratia nuclease through efficient secretory fusion expression and recombination in methylotrophic yeast
  • Method for preparing serratia nuclease through efficient secretory fusion expression and recombination in methylotrophic yeast
  • Method for preparing serratia nuclease through efficient secretory fusion expression and recombination in methylotrophic yeast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Preparation of prodigal nuclease based on HV1 fusion protein

[0037] (1) Construction of fusion vector pPICZαA-HV1-NUC

[0038] ①Fusion protein sequence design

[0039] In this example, the N-terminus of the fusion protein involved is the HV1 sequence (SEQ ID No: 1), the middle is the enterokinase cleavage site sequence (DDDDK), and the C-terminus is the prodigal nuclease (SEQ ID No: 4 ). The complete sequence of the fusion protein (SEQ ID No: 5) is:

[0040] VVYTDCTESGQNLCLCEGSNVCGQGNKCILGSDGEKNQCVTGEGTPKPQSHNDGDFEEIP

[0041]

[0042] The underlined part is the amino acid sequence of the HV1 mutant, the double underlined part is the amino acid sequence of the enterokinase cleavage site, and the underlined part is the NUC amino acid sequence.

[0043] ②Fusion gene design and synthesis

[0044] According to the above amino acid sequence, the nucleotide sequence was designed according to the codon preference of Pichia pastoris. Design a stop codon ...

Embodiment 2

[0057] Example 2. Preparation of prodigal nuclease based on HV2 fusion protein

[0058] (1) Construction of fusion vector pPIC9K-HV2-NUC

[0059] ①Fusion protein sequence design

[0060] In this example, the N-terminus of the fusion protein involved is the HV2 sequence (SEQ ID No: 2), the middle is the enterokinase cleavage site sequence (DDDDK), and the C-terminus is the prodigal nuclease (SEQ ID No: 4 ). The complete sequence of the fusion protein (SEQ ID No: 7) is:

[0061] VVYTDCTESGQNLCLCEGSNVCGQGNKCILGSDGEKNQCVTGEGTPKPQSHNDGDFEEIP

[0062]

[0063] The underlined part is the amino acid sequence of the HV2 mutant, the double underlined part is the amino acid sequence of the enterokinase cleavage site, and the underlined part is the NUC amino acid sequence.

[0064] ②Fusion gene design and synthesis

[0065] According to the above amino acid sequence, the nucleotide sequence was designed according to the codon preference of Pichia pastoris. Design a stop codon s...

Embodiment 3

[0078] Example 3. Preparation of non-hirudin fusion protein by prodigal nuclease

[0079] (1) Construction of vector pPICZαA-NUC

[0080] ①Protein sequence design

[0081] In this embodiment, the amino acid sequence of Prodigobacterium nuclease NUC (SEQ ID No: 4) is:

[0082]

[0083] ② Gene design and synthesis

[0084] According to the above amino acid sequence, the nucleotide sequence was designed according to the codon preference of Pichia pastoris. Design a stop codon sequence behind the expression sequence, and design XhoI and NotI restriction enzyme sites at the N-terminal and C-terminal of the sequence, respectively, and entrust the whole gene synthesis.

[0085] ③The NUC sequence synthesized by the whole gene was double-digested with XhoI and NotI and then inserted into the pPICZαA vector that had been digested with the same enzyme and transformed into Escherichia coli DH5α or Top10 cloning host bacteria. Positive clones were picked, and the recombinant plasmid ...

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Abstract

The invention discloses a method for preparing serratia nuclease through efficient secretory fusion expression and recombination in methylotrophic yeast. Hirudin is located at the N terminal of the fusion expression, nuclease is located at the C terminal of the fusion expression, the middle is a DDDDK sequence, and the specific structure is shown as follows: Hirudin-DDDDK-NUC. According to the method, the serratia nuclease with high yield, high specific activity and low cost is obtained by utilizing the closed activity of the hirudin and the high expression of the hirudin for the first time.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for efficiently secreting, fused, expressing, and recombiningly preparing prodigal nuclease in methanolic yeast. Background technique [0002] The enzyme capable of cutting the phosphodiester bond of the polynucleotide chain is called nuclease (NUC for short), which belongs to hydrolase and acts on the P-O position of the phosphodiester bond. Prodigy nuclease is a non-limiting broad-spectrum nuclease derived from Serratia marcescens (also known as prodigy), which can cut between any nucleotides in the nucleic acid chain, and can cut any form Nucleic acid (single-stranded, double-stranded, linear, circular or supercoiled form of DNA or RNA). The enzymatic digestion efficiency of Prodigobacterium nuclease is much higher than that of other nucleases, and its activity is 34 times that of bovine pancreatic DNase I and 6 times that of Staphylococcus aureus nuclease. T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N1/19C12N15/81C12R1/645
CPCC12N9/22C07K14/815C12N15/815C07K2319/00
Inventor 张淳张增涛姜亮赵志龙史晓委张海娟
Owner 山东仁瑞生物科技有限公司
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