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Pyrrolysyl-tRNA synthetase mutant and application thereof

A technology of pyrrolysyl and synthetase, which is applied in the field of enzyme engineering, can solve the problems of low efficiency of unnatural amino acids, achieve the effects of improving introduction efficiency, increasing recognition rate, and facilitating popularization and application

Pending Publication Date: 2022-05-10
ZHEJIANG UNIV HANGZHOU GLOBAL SCI & TECH INNOVATION CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problem of low efficiency of introducing unnatural amino acids, the present invention provides a pyrrolysyl-tRNA synthetase (PylRS) mutant transformed by site-directed mutagenesis, which can efficiently introduce multiple non-natural amino acids into target proteins. natural amino acid

Method used

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  • Pyrrolysyl-tRNA synthetase mutant and application thereof
  • Pyrrolysyl-tRNA synthetase mutant and application thereof
  • Pyrrolysyl-tRNA synthetase mutant and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Construction of MbPylRS (N311A / C313A) and activity comparison with MmPylRS (N346A / C348A)

[0035] MmPylRS comprises 454 amino acid residues and has 74% homology with MbPylRS. It is reported that MmPy lRS(N346A / C348A) has high substrate procrastination and can accept more than 40 unnatural amino acids. Based on the sequence comparison between MmPylRS and MbPylRS, the present invention uses site-directed mutagenesis to construct pBK-MbPylRS (N311A / C313A), and detects the expression of green fluorescent protein (superfolder green fluorescent protein, sf GFP) on 15 different benzene The alanine analog unnatural amino acid was tested for introducing efficiency. The expression vector pGFP2TAG was obtained by using the pGFP vector as a template to mutate the serine triplet base sequence at the second site of the GFP protein into TAG by PCR.

[0036] The activity detection method is as follows:

[0037] 1. Plasmids pBK-MbPylRS (N311A / C313A) and pBK-MmPylRS (N346A / C3...

Embodiment 2

[0045] Example 2 Construction and activity detection of MbPylRS (V31I, T56P, A100E, N311A, C313A) mutants

[0046] PylRS mainly contains two functional domains, the C-terminal catalytic domain consisting of about 250 amino acid residues, and the N-terminal tRNA binding domain consisting of about 140 amino acid residues. The present invention uses MbPylRS (N311A / C313A) as a template to introduce a series of mutations at the N-terminal of PylRS to further improve its catalytic efficiency.

[0047] 1. Construction of single point mutants

[0048] Design mutation primers according to the nucleotide sequence of MbPylRS (SEQ ID NO.2), using fast PCR technology, using the recombinant vector pBK-MbPylRS (N311A / C313A) as a template;

[0049] (1) A single mutation is introduced at position 31 of the amino acid sequence of MbPylRS, and the primers are:

[0050] Forward primer 1: 5'-catgaa att agccgcagcaaaatctatattga-3' (the underline is the mutated base)

[0051] Reverse primer 2: 5...

Embodiment 3

[0080] Example 3 Expression and purification of GFP protein introducing unnatural amino acid

[0081] The present invention utilizes MbPylRS (V31I / T56P / A100E / N311A / C313A) to introduce an unnatural amino acid into the second amino acid site of the GFP protein, and induces the expression to purify the GFP protein introduced with the unnatural amino acid ( image 3 ), providing broad prospects for expanding the application of unnatural amino acids in the field of enzyme engineering.

[0082] The expression purification method is as follows:

[0083] 1. Plasmids pBK-MbPylRS (V31I / T56P / A100E / N311A / C313A) and pGFP2TAG were co-transformed into E. coli transformation-competent DH10B; +50μg / mL kanamycin resistance plate; 37 ℃ inverted culture overnight;

[0084] 2. Pick a single colony into 5mL LB medium containing KT resistance, culture overnight at 37°C and 220rpm;

[0085] 3. The next day, transfer 1% transfer volume to 400mL LB liquid medium, culture at 37°C 220rpm for 3h, add a...

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Abstract

The invention discloses a pyrrole lysyl-tRNA synthetase mutant and application thereof. The mutant is obtained by mutating an amino acid sequence shown in SEQ ID NO.3 at a 31st site, a 56th site, a 100th site, a 311th site and a 313th site. According to the invention, the MbPylRS sequence is modified, so that the tRNA recognition rate is improved, and the introduction efficiency of unnatural amino acid is further improved. According to verification, the combination efficiency of mutation of different non-natural amino acids MbPylRS is higher than that of MmPylRS, the introduction efficiency of the modified MbPylRS to ten non-natural amino acids is improved by 3-6 times compared with that of MbPylRS (N311A / C313A), and the non-natural amino acids are successfully used for expression of GFP protein. The method solves the problem of low introduction efficiency of non-natural amino acid, and is convenient to popularize and apply.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to pyrrolysyl-tRNA synthetase mutants and applications thereof. Background technique [0002] Enzyme, as a specific and efficient biocatalyst, has been widely used in medicine, analysis and detection, industrial and agricultural production and other fields. Through site-directed mutation and directed evolution, enzymes can be modified to meet the needs of production and life. However, there are only 20 natural amino acids encoded by the genetic code, which limits the expansion of the structure and function of enzymes. Pyrrolysyl-tRNA synthetase (PylRS) and its homologous tRNA Pyl It is an ideal genetic code expansion tool. Different mutants of this enzyme can integrate more than 100 unnatural amino acids (UAAs) into proteins, making it possible for unnatural amino acids to be used in the modification of enzyme molecules. [0003] One of the goals of enzyme engineering ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/70
CPCC12N9/93C12Y601/01026C12N15/70C07K14/43595
Inventor 于浩然马爽
Owner ZHEJIANG UNIV HANGZHOU GLOBAL SCI & TECH INNOVATION CENT
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