Transcription regulatory protein msir mutant protein of Rhizobium tianshanensis and its application in canavanine biosensor

A technique for mutating proteins and transcriptional regulation, applied in the field of bioengineering, can solve undiscovered problems and achieve the effect of improving regulatory activity and affinity

Active Publication Date: 2021-12-14
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Through extensive and in-depth research, the inventors screened out the key amino acid sites that can significantly improve the transcriptional regulatory activity of the MsiR mutant protein on canavanine through a large number of screenings. The inventors found that the wild-type MsiR mutant protein After the modification of the key site, the fluorescence value of the transcriptional regulatory protein MsiR mutant protein in response to canavanine was twice as high as that of the wild type, and the ITC results showed that the mutation greatly increased the affinity of the MsiR protein to canavanine. Through the search, no patent publications related to the patent application of the present invention have been found

Method used

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  • Transcription regulatory protein msir mutant protein of Rhizobium tianshanensis and its application in canavanine biosensor
  • Transcription regulatory protein msir mutant protein of Rhizobium tianshanensis and its application in canavanine biosensor
  • Transcription regulatory protein msir mutant protein of Rhizobium tianshanensis and its application in canavanine biosensor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the preparation of transcription regulator protein MsiR (MsiR1-297aa) and MsiR-CTD (MsiR83-297aa) recombinant expression plasmid and recombinant expression transformant

[0033] (1) MsiR-CTD (MsiR83-297aa) is the C-terminal of the transcriptional regulatory protein MsiR. After the protein gene sequence of MsiR-CTD (MsiR83-297aa) is synthesized, the protein gene sequence is connected to the pET 21b empty vector (that is, the Carrier), at the same time with restriction endonucleases BamHI and NdeI double-digested overnight, then purified by agarose gel electrophoresis, DNA kit recovery, the recovered target fragment and the empty vector pET 21b were ligated in T4 DNA Under the action of the enzyme, connect at 4°C for 12 hours to obtain the recombinant plasmid pET21b-msiR-CTD, which is further transformed into competent cells BL21(DE3) to pick positive clones to obtain the recombinant expression transformant E.coli BL21(DE3) / pET21b-msiR-CTD.

[0034](2) Msi...

Embodiment 2

[0035] Example 2: Construction of a screening system for the activity of the transcriptional regulatory protein MsiR on canavanine

[0036] Using the msiR gene knockout strain of Bradyrhizobium in Tianshan TC4 as the starting strain, the PTCV141 plasmid and PTM5 plasmid were introduced into Tianshan Rhizobium (host cells) to construct a screening strain TC4 (PTCV141 plasmid and PTM5 plasmid), in which PTCV141 is a vector for constructing msiR gene mutation; PTM5 is to clone mCherry gene into the downstream of the promoter PmsiA regulated by MsiR protein, and it is used to quantitatively detect the influence of different MsiR mutant proteins on the transcriptional regulation of PmsiA and the expression of msiA promoter Expressed by the ratio of mCherry fluorescence value to OD600nm.

Embodiment 3

[0037] Embodiment 3: regulatory protein MsiR and MsiR-CTD mutant construction

[0038] The protein homology structure comparison was carried out through the NCBI database, and the 3FD3_A with the highest similarity in the protein structure database was selected as the template for homology modeling. The structural model of MsiR was built using DS Modeling2.5, and the 10.0 Å in the substrate binding pocket was selected. Amino acid, then carry out molecular docking with L-canavanine small molecule, and screen out 10 mutant amino acids that affect substrate binding through the conservation of amino acid sequence and docking scoring, and perform alanine mutation scanning. The primer sequences were designed for the mutated amino acids. The primer sequences are shown in Table 1. Using pET21b-msiR-CTD and PTCV141 as templates, one-step PCR was used, and high-fidelity polymerase was used for PCR. The PCR reaction conditions were as follows: PCR reaction system with a total volume of 50...

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Abstract

The present invention provides a MsiR mutant protein of the Rhizobium tianshanensis transcriptional regulatory protein and its application in a canavanine biosensor. The mutant protein is a non-natural protein, and the mutant protein has the ability to specifically respond to canavanine. The mutant protein is mutated in a core amino acid related to canavanine binding in the wild-type MsiR: the 133rd position is mutated from aspartic acid (D) to alanine (A). The protein sequence of the mutant protein is shown in SEQ As shown in ID NO.1, the nucleotide sequence is as SEQ ID NO.2. Compared with the wild type, the fluorescence value of the mutant protein in response to canavanine is increased by 2 times, and the MsiR mutant protein C-terminal effector The binding affinity of the binding region to canavanine is increased by 1.5 times; at the same time, the regulatory activity of the MsiR transcriptional regulatory protein is improved by screening the mutant protein of the protein.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to the MsiR mutant protein of the transcription regulation protein of Rhizobium tianshanensis and its application in a canavanine biosensor. Background technique [0002] Canavanine is an unnatural amino acid isolated from canavalin, which is widely found in legumes and their seeds. The structure of canavanine is similar to that of arginine, so it will cause canavanine to replace arginine Synthesis of protein, canavanine will be wrongly incorporated into the newly synthesized protein during protein synthesis, interfere with the normal metabolic reaction of RNA and DNA, affect the normal protein synthesis and the normal metabolism of arginine, and can be used in agriculture and It is widely used in the pharmaceutical industry. Due to the difference in the structure of canavanine and arginine, the function of the formed protein will also be abnormal, resulting in pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/195C12N15/31C12N15/70C12N15/74C12N15/65C12N1/21C12Q1/02C12Q1/6897C12R1/41C12R1/19
Inventor 马延和蔡韬张洁赵亚琪夏海容王钦宏
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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