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An Orthogonal Aminoacyl-tRNA Synthetase/tRNA System Using a Chimeric Design Approach

A design method, a technology for synthesizing enzymes, applied in the field of chemical biology, which can solve the problems of increasing restrictions, not widely orthogonal, and low activity of unnatural amino acids

Active Publication Date: 2021-03-16
杭州嵌化合生医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Pyrrolysyl—tRNA synthetase (PylRS) / tRNA CUA Although the orthogonal system has successfully introduced about 100 unnatural amino acids into bacteria and eukaryotic cells, it has the following problems: 1. The activity of some unnatural amino acids is very low, resulting in high experimental costs , the signal-to-noise ratio is low; 2. These introduced unnatural amino acids are derivatives of lysine and phenylalanine, and it is difficult to introduce amino acids with other chemical structures
However, there are many problems in this way: 1. For other aminoacyl tRNA synthetase / tRNA systems, the anticodon-binding domain of aminoacyl tRNA synthetase needs to be evolved so that it can recognize unassigned codons (stop codons) , rare codons and quadruple codons); 2, this system does not have universal orthogonality, the source and bacteria can be used in eukaryotic cells, and the source of eukaryotes can be used in bacteria, which is Added many restrictions to our application
Although the above solutions have overcome the problems in the field of genetic code extension to a certain extent, they have not fundamentally solved the problem of non-extensive orthogonality of natural amino acid aminoacyl tRNA synthetases.

Method used

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  • An Orthogonal Aminoacyl-tRNA Synthetase/tRNA System Using a Chimeric Design Approach
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  • An Orthogonal Aminoacyl-tRNA Synthetase/tRNA System Using a Chimeric Design Approach

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Experimental program
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Embodiment 1

[0065] Embodiment 1: Construction of chimeric histidine tRNA

[0066] In the present invention, the chimeric tRNA and the reporter gene GFP190TAG-His6 are constructed on the same plasmid, controlled by the lpp promoter and the pBAD promoter respectively. The specific construction method is as follows:

[0067] (1) Construct GFP190TAG-His6 onto the pNEG vector

[0068] The sequence of GFP190TAG-His6 is shown in SEQ ID No.1. Primers pNEG-gfp-F and pNEG-gfp-R were designed to amplify using the existing plasmid containing this gene as a template, and pNEG-gfp-v-F and pNEG-gfp-v-F were simultaneously designed and synthesized. pNEG-gfp-v-R, using the previous pNEG as a template to amplify the vector. After the agarose gel was recovered, the Gibson assembly was used to transform DH10B competent cells, and the single clone was selected and sequenced to obtain the plasmid pNEG-GFP190TAG-His6;

[0069](2) Design chimeric histidine tRNA and clone into pNEG-GFP190TAG-His6 vector

[00...

Embodiment 2

[0073] Embodiment 2: Construction of chimeric histidyl-tRNA synthetase

[0074] The chimeric histidyl-tRNA synthetase is composed of two parts, one is the pyrrolysyl-tRNA synthetase tRNA binding domain, and the other is the catalytic domain of the histidyl-tRNA synthetase, see figure 2 and 3 c.

[0075] The construction of the chimeric histidyl-tRNA synthetase includes the selection of the tRNA binding domain of the pyrrolysyl-tRNA synthetase, the selection of the catalytic domain of the histidyl-tRNA synthetase, and the selection of the fusion mode of the two.

[0076] (1) Analysis and selection of the tRNA binding domain of pyrrolysyl-tRNA synthetase

[0077] In the present invention, it is necessary to first clone the pyrrolysyl-tRNA synthetase tRNA binding domain onto the pBK carrier. This tRNA binding domain can be divided into two subdomains, and the N-terminal 1-149 recognizes the variable region of the corresponding tRNA. And the T loop, the 185-240 section recogni...

Embodiment 3

[0084] Example 3: Optimization of chimeric histidyl-tRNA synthetase linker peptide

[0085] In the construction of chimeric protein, the connecting peptide between two chimeric fragments is crucial to the activity of chimeric molecule, in the present invention we have tested the length of connecting peptide and the kind of connecting peptide ( Figure 11 ).

[0086] (1) Optimization of connecting peptide length

[0087] The length of the designed flexible connecting peptide (GS-type) is 0, 6, 12, 18 and 24 amino acids, named chHisRS4-2, -3, -4, -5 and -6, respectively, and the nucleotide sequence of the corresponding connecting peptide See Table 3-1. At the same time, the N-terminal 15 amino acids of histidine-tRNA synthetase were deleted on the basis of the 0 amino acid linking peptide, and named chHisRS4-1. The designed primers were constructed on the vector by the Q5 site-directed mutagenesis kit (NEB), corresponding The primers are listed in Table 3-2.

[0088] (2) Con...

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Abstract

Provided is construction of an orthogonal aminoacyl-tRNA synthetase / tRNA system by using a chimeric design method. The system transplants the universal orthogonality of eukaryotes and prokaryotes of a pyrrolysyl-tRNA synthetase / tRNA system by means of rational chimeric design, includes several chimeric tRNA synthetase / tRNA systems, such as histidine, phenylalanine, alanine and serine systems, and can be applied to prokaryotes and eukaryotes, as well as orthogonal transformation of other aminoacyl-tRNA synthetase / tRNA pairs.

Description

technical field [0001] The invention belongs to the technical field of chemical biology, and in particular relates to an orthogonal aminoacyl-tRNA synthetase / tRNA system constructed by a chimeric design method. Background technique [0002] Protein is the main substance that performs functions in organisms, and 20 amino acids encoded by 61 codons are synthesized in ribosomes. Although the 20 kinds of amino acids endow proteins with the characteristics of participating in various physiological and biochemical activities, only a few active groups such as sulfhydryl groups and hydroxyl groups can be chemically manipulated among the 20 kinds of amino acids. In order to better study the physiological functions of proteins, the genetic code extension technology that can introduce other unnatural amino acids with active groups has emerged as the times require. Genetic code expansion technology utilizes the orthogonal aminoacyl tRNA synthetase / tRNA system to identify unnatural amin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N15/85C12N15/70
CPCC12N9/93C12N15/70C12N15/85
Inventor 林世贤柳光龙丁文龙赵红霞
Owner 杭州嵌化合生医药科技有限公司