CN112661820B

CN112661820B - Rhizobium tianshanense transcription regulation protein MsiR mutant protein and application thereof in canavanine biosensor

Info

Publication number: CN112661820B
Application number: CN202011637703.7A
Authority: CN
Inventors: 马延和; 蔡韬; 张洁; 赵亚琪; 夏海容; 王钦宏
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-12-14
Anticipated expiration: 2040-12-31
Also published as: CN112661820A

Abstract

The invention provides a rhizobium tiansiense transcription regulation protein MsiR mutant protein and application thereof in a canavanine biosensor, wherein the mutant protein is an unnatural protein and has the capability of specifically responding to canavanine, and the mutant protein is mutated in 1 core amino acid related to combination of the wild MsiR and the canavanine: the 133 th site is mutated from aspartic acid (D) to alanine (A), the protein sequence of the mutant protein is shown as SEQ ID NO.1, the nucleotide sequence is shown as SEQ ID NO.2, the mutant protein of the invention is improved by 2 times compared with the wild type fluorescence value responding to canavanine, and the binding affinity of the MsiR mutant protein C-terminal effector binding region and the canavanine is improved by 1.5 times; meanwhile, the regulation activity of the MsiR transcription regulation protein is improved by screening the mutant protein of the protein.

Description

Rhizobium tianshanense transcription regulation protein MsiR mutant protein and application thereof in canavanine biosensor

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a tianshan rhizobium transcription regulation protein MsiR mutant protein and application thereof in a canavanine biosensor.

Background

Canavanine is an unnatural amino acid isolated from canavanine and widely exists in leguminous plants and seeds thereof, the structure of the canavanine is similar to arginine, so that the canavanine can replace arginine to synthesize protein, the canavanine can be mistakenly doped into newly synthesized protein in the protein synthesis process, the normal metabolic reaction of RNA and DNA is interfered, the synthesis of normal protein and the normal metabolism of arginine are influenced, the canavanine can be widely applied to the agricultural and pharmaceutical industries, and due to the difference of the structures of the canavanine and the arginine, the function of the formed protein is abnormal, the protein is inactivated, and the anabolic activity is realized, so that the canavanine is very important for quickly detecting the canavanine.

The slow-growing type Tianshan rhizobia can symbiotically nodulate and fix nitrogen at the root of the liquorice, and provide a nitrogen source required by plant growth. The establishment of such symbiotic relationships relies on a complex communication between rhizobia and host plants, where the MsiAR system, an export system of canavanine, is present in the rhizobium of Tianshan, and is capable of specifically recognizing intracellular canavanine, and the MsiR protein is capable of initiatingmsiAExpression of the gene in turn secretes intracellular canavanine to the outside of the cell, which acts as a co-inducer.

Leguminous plants have long evolved to use canavanine as a "biochemical weapon" for protection against herbivorous insects and animals, as well as against harmful microorganisms. Related researches also show that the canavanine has anti-tumor activity, and the development of a method for quickly and efficiently detecting the canavanine has important value for researching the physiological function, the synthetic mechanism and the anti-cancer mechanism of the canavanine. The currently reported detection methods of L-canavanine include a chemical spectrophotometer detection method, a high performance liquid chromatography method, a capillary electrophoresis method, an amino acid analysis method, a nuclear magnetic resonance detection method and the like, but the methods have the defects of complex pretreatment, low sensitivity, low specificity, complex operation, long time consumption, low flux and the like.

The Transcription regulation Factor (Transcription Factor) is an important regulation protein in a microorganism body, a specific metabolite is used as a ligand, downstream genes are regulated after the ligand is combined with a promoter region of the regulated protein, and the amount of the required ligand has positive correlation with the regulation effect in a certain range. Based on the above principle, the transcription regulatory factor in the microorganism may be designed as a Biosensor (Biosensor) capable of recognizing a specific target metabolite, thereby enabling rapid detection of the target metabolite. Compared with the traditional rational design, the method has the advantages of high detection speed, high sensitivity and the like, and related reports of the L-canavanine single-cell biosensor do not exist at present, so that a real-time, accurate and sensitive canavanine detection method is urgently needed.

Through extensive and intensive research, the inventor screens out key amino acid sites capable of remarkably improving the transcriptional regulation activity of the MsiR mutant protein on canavanine through a large amount of screening, the inventor finds that after the key sites in the wild-type MsiR mutant protein are modified, the fluorescence value of the transcriptional regulation protein MsiR mutant protein responding to the canavanine is improved by two times compared with the wild-type MsiR mutant protein, and an ITC result shows that the mutation greatly improves the affinity of the MsiR protein and the canavanine, and through retrieval, a patent publication document related to the patent application is not found.

Disclosure of Invention

Aiming at the defects of the prior art, the invention is based on the transcription regulation and control factor MsiR, the concentration of L-canavanine is coupled with the fluorescence intensity, a biosensor capable of detecting the concentration of L-canavanine is constructed, the specificity quantitative detection of the L-canavanine can be realized, and the regulation and control activity of the MsiR transcription regulation and control protein is improved by screening the mutant protein of the protein.

One of the objectives of the present invention is to provide a rhizobium tiansiense transcriptional regulatory protein MsiR mutein, wherein the mutein is a non-natural protein and has the ability to specifically respond to canavanine, and the mutein undergoes mutation in 1 core amino acid of wild-type MsiR associated with canavanine binding: aspartic acid (D) is mutated into alanine (A) at position 133, the protein sequence of the mutant protein is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2.

Further, the transcription regulation protein MsiR is derived from bradyrhizobium japonicum in Tianshan mountain.

The other object of the present invention is to provide a polynucleotide, wherein the vector comprises the polynucleotide of claim 3, wherein the C-terminal nucleotide sequence encoded by the polynucleotide is shown in SEQ ID NO.3, and the C-terminal protein sequence encoded by the polynucleotide is shown in SEQ ID NO. 4.

Another object of the present invention is to provide two vectors comprising the polynucleotide of claim 3

The fourth object of the present invention is to provide two host cells comprising the vector of claim 4 or having the polynucleotide of claim 3 integrated into its genome.

The fifth object of the present invention is to provide a method for producing a mutein, comprising culturing the host cell of claim 5 under suitable conditions for expression to express the transcriptional regulator protein MsiR mutein; and/or isolating the transcriptional regulatory protein MsiR mutein.

The sixth purpose of the invention is to provide a method for constructing a canavanine single-cell biosensor, which comprises the following steps:

(1) based on the transcription regulation factor MsiR, by using slow rooting rhizomatous bacteria in Tianshan mountainmsiRConstructing PTCV141 and PTM5 plasmids by taking the gene knockout strain as a starting strain, wherein the PTCV141 is used for constructingmsiRA vector for gene mutation; PTM5 is obtained by cloning mCherry gene to the downstream of promoter Pmsia regulated by Msry protein, and is used for quantitatively detecting the influence of different Msry mutant proteins on Pmsia transcription regulation, so as to construct a screening strain TC4M302, wherein the expression condition of the msiA promoter is represented by the ratio of mCherry fluorescence value to OD600 nm;

(2) the constructed single-cell biosensor with coupled fluorescence signals and canavanine concentration detects the linear range of the single-cell biosensor, and the fluorescence signal intensity and the canavanine concentration have good linear relation under the canavanine concentration of 0 mmol/L to 5.68 mmol/L.

Furthermore, the canavanine single-cell biosensor consists of a transcription regulatory factor MsiR coding gene of the slow rhizobium in the source, a promoter region and a terminator region of the gene msiA in the slow rhizobium in the source, a red fluorescent protein coding gene mCherry and a plasmid skeleton pYC12 plasmid.

The seventh object of the present invention is to provide the use of the mutein of claim 1 for the construction of a biosensor in response to canavanine or for the detection of canavanine in vitro.

Further, the single-cell biosensor for detecting the concentration of L-canavanine comprises the following steps:

(1) pre-culturing rhizobium japonicum containing PTCV141 plasmid and PTM5 plasmid at 28 ℃ for more than 12 hours until entering stationary phase;

(2) inoculating the pre-cultured rhizobium tiansiense into a fresh TY culture medium containing L-canavanine with different concentrations in an inoculation amount of 1: 50;

(3) and (5) when the bacterial liquid OD600 is between 0.6 and 0.8, culturing for 48 to 72 hours, and then measuring the fluorescence intensity.

Compared with the prior art, the invention has the advantages and positive effects that: the mutein is prepared by transcriptional regulation protein MsiR of rhizobium tianschanicum from a wild type, amino acid at position 133 is mutated into alanine from aspartic acid (D), a biosensor capable of detecting the concentration of L-canavanine is constructed based on the transcriptional regulation factor MsiR, the specificity quantitative detection of the L-canavanine can be realized, the mutein is improved by 2 times compared with the fluorescent value of the response of the wild type to the canavanine, and the affinity of the combination of the C-end effector binding region of the MsiR mutein and the canavanine is improved by 1.5 times; meanwhile, the regulation activity of the MsiR transcription regulation protein is improved by screening the mutant protein of the protein.

Drawings

FIG. 1 is a schematic diagram of a single-cell biosensor for MsiR muteins according to the present invention;

FIG. 2 is an SDS-PAGE electropherogram of purified MsiR muteins according to the present invention;

FIG. 3 shows the expression of the msiR mutein msi A promoter according to the invention;

FIG. 4 shows the results of an isothermal titration calorimetry assay of the MsiR mutein MsiR (D133A) -CTD according to the invention with canavanine;

FIG. 5 is an experimental result of an isothermal titration calorimetry experimental determination of MsiR-CTD and canavanine according to the present invention;

FIG. 6 shows the results of isothermal titration calorimetry of the negative control buffer and canavanine of the present invention.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

Example 1: preparation of recombinant expression plasmids and recombinant expression transformants of transcriptional regulatory proteins MsiR (MsiR1-297aa) and MsiR-CTD (MsiR83-297aa)

(1) MsiR-CTD (MsiR83-297aa) is the C end of a transcriptional regulatory protein MsiR, after the protein gene sequence of the MsiR-CTD (MsiR83-297aa) is synthesized, the protein gene sequence is connected to a pET21b empty vector (namely the vector), restriction enzymes BamHI and NdeI are used for double digestion overnight, then agarose gel electrophoresis purification and DNA kit recovery are carried out, the recovered digestion target fragment and the empty vector pET21b are connected for 12 hours at 4 ℃ under the action of T4 DNA ligase to obtain a recombinant plasmid pET21b-msiR-CTD, and the recombinant plasmid pET 21-b-msiR-CTD is further transformed into a competent cell BL21(DE3) to pick up a positive clone, namely, the recombinant expression transformant E, coli BL21(DE 3)/pET 21b-msiR-CTD is obtained.

(2) MsiR (MsiR1-297aa) is a complete sequence of a transcription regulatory protein MsiR, after the protein gene sequence of the MsiR (MsiR1-297aa) is synthesized, the protein gene sequence is connected to a pYC12 plasmid empty vector (namely the vector), restriction enzymes Hind3 and KpnI are used for double digestion overnight, and then agarose gel electrophoresis purification and DNA kit recovery are carried out,connecting the recovered enzyme digestion target fragment and an empty vector pYC12 plasmid at 4 ℃ for 12 hours under the action of T4 DNA ligase to obtain a recombinant plasmid pYC12 plasmid-msiR, and then converting the recombinant plasmid pYC12 plasmid-msiR into the recombinant plasmid pYC12 plasmid-msiRBW20767Coli (i.e., host cell) and selecting positive clone to obtain recombinant expression transformant E. coli BW 20767/pYC 12-msiR, i.e., PTCV 141.

Example 2: construction of transcription regulation protein MsiR for activity screening system of canavanine

Taking an msiR gene knockout strain of bradyrhizobium in TC4 Tianshan as an initial strain, introducing PTCV141 plasmid and PTM5 plasmid into Tianshan rhizobium (namely host cells) to construct a screened strain TC4 (PTCV 141 plasmid and PTM5 plasmid), wherein PTCV141 is a vector for constructing msiR gene mutation; PTM5 is obtained by cloning mCherry gene to the downstream of promoter PmsIA regulated by MsriR protein for quantitatively detecting the influence of different MsriR mutant proteins on PmsiA transcription regulation, and the expression condition of the msiA promoter is represented by the ratio of mCherry fluorescence value to OD600 nm.

Example 3: construction of regulatory protein MsiR and MsiR-CTD mutant

Carrying out comparison of protein homologous structures through an NCBI database, selecting 3FD3_ A with the highest similarity in the protein structure database as a template for homologous modeling, constructing a MsiR structure model by using DS Modeling2.5, selecting 10.0 amino acids in a substrate binding pocket, then carrying out molecular docking with L-canavanine small molecules, screening 10 mutant amino acids influencing substrate binding through the conservatism of amino acid sequences and the docking and typing condition, carrying out alanine mutation scanning, designing primer sequences aiming at the 10 mutant amino acids, wherein the primer sequences are shown in Table 1, carrying out PCR by using pET21b-msiR-CTD and PTCV141 as templates respectively and adopting a one-step method PCR, carrying out PCR by using high-fidelity polymerase, and the PCR reaction conditions are as follows: in a PCR reaction system with a total volume of 50. mu.L, 50-100 ng of template, 5. mu.l (2mM) of deoxyribonucleoside triphosphate (dNTP), 10. mu.l of 5 Xfast polymerase buffer, 1. mu.L of primer-F (10. mu.M), 1. mu.L of primer-R (10. mu.M) and 1. mu.L of FastPfu DNA polymerase were added, and sterilized distilled water was added to 50. mu.L. The PCR reaction program was set as: 2 min at 98 ℃ (30 s at 98 ℃, 30s at 58 ℃, 3min at 72 ℃), 35 cycles, 10 min at 72 ℃, and the product was stored at 4 ℃.

The PCR product was analyzed and verified by agarose gel electrophoresis, and digested for 4h at 37 ℃ with the addition of the restriction enzyme DpnI. Transfer of the digests into E. coliBL21(DE3) competent cells andBW20767and E.coli, respectively coating the Escherichia coli in plates containing ampicillin and gentamicin, placing the plates in an incubator at 37 ℃ for standing culture for about 12 hours, picking the obtained monoclonal colonies into a test tube containing 4ml of LB culture medium for culture, and sequencing corresponding genes.

TABLE 1

The mutant protein is mutated in 1 core amino acid of a wild-type transcription regulatory protein MsiR which is related to the binding of canavanine.

Example 4: construction of a mutant library of regulatory proteins MsiR

Constructing a 133-amino acid saturation mutant library of MsiR with enhanced transcriptional regulation activity according to the mutant protein alanine scanning result, and designing a mutation primer by adopting degenerate codon NNK, wherein the mutation primer comprises the following steps:

the upstream primer is as follows: 133-F: 5 'GGCTACGGTCTCgcggtatgNNKctggtcgtcgacggcgacgttca 3'

The downstream primer is: 133-R: 5 'GGCTACGGTCTCtaccgccgaatggctgcagcgcgga 3'

The PCR system and procedure were the same as in example 3, and the obtained monoclonal colonies were ligated with M.tianshanense, which had been transformed with PTM5 plasmid, to obtain a zygote, and the ligated colonies were picked up in a 96-well deep-well plate for culture, and the expressed proteins were screened for high-throughput regulatory activity, and the mutants with fluorescence values 2-fold higher than the wild type were subjected to corresponding gene sequencing.

Example 5: detection of regulatory Activity of mutant Strain

Selecting a mutant strain with improved regulation activity, selecting three clones of the mutant strain, a positive control wild strain 141TC4 and a negative control 12TC4 strain, inoculating the three clones into a 96-well plate, culturing for 4 days at 28 ℃ in a TY liquid culture medium, then inoculating the clones into TY liquid culture medium with L-canavanine concentrations of 0.1 ug/ml,10 ug/ml,100 ug/ml and 1000 ug/m respectively, wherein the inoculation ratio of the seed liquid is 1:100, when the bacterial liquid OD600 is 0.6-0.8, the mutant strain is cultured for 4 days at 28 ℃ to measure the fluorescence intensity, and the mCherry fluorescence value and the OD600 value are detected by an enzyme labeling instrument, and a multifunctional detector of a biotechnology company, the model of which is Synergy NEO2, is used, and the fluorescence intensity of the mutant strain is improved by more than 2 times compared with the positive control wild strain 141TC4 and the negative control 12TC4 according to be seen in figure 3.

Example 6: inducible expression and purification of regulatory protein MsiR-CTD (MsiR83-297aa) mutant

Preparing 50mL of seed liquid, wherein the culture medium is LB liquid culture medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L), picking single colony of the genetically engineered bacteria by using an inoculating loop, inoculating into the culture medium, and culturing at 37 ℃ and 200rpm overnight. Transferring the seed liquid cultured overnight to a fermentation medium (LB medium) with the inoculation amount of 1%, culturing at 37 ℃ and 200rpm until the A is 6000.6-1.0, adding 0.5 mM IPTG, and inducing at 20 ℃ and 200rpm for 10-12 h. The thalli is collected by centrifugation at the temperature of 4 ℃ and the rpm of 5500, washed twice by sodium phosphate buffer solution (100mM, pH 7.0), crushed by a high-pressure homogenizer, centrifuged at 13000rpm to leave supernatant, purified and recovered by a metal affinity chromatography (nickel column) method, and the target protein is subjected to a desalting method to remove imidazole to obtain MsiR-CTD (MsiR83-297aa) mutant pure enzyme solution, and the result shows that the method can obtain a relatively pure protein mutant, and the purified protein has a single band, the molecular weight is 25KD and the purity is more than 95%.

Example 7: regulation protein MsiR mutant and determination of affinity of MsiR wild type C-terminal effector binding region and canavanine thereof

Isothermal titration calorimetry experiments were conducted at 25 ℃ using a MicroCal Auto-iTC200 isothermal titration calorimeter (GE Healthcare Life Sciences, USA). In the experiment, purified MsiR-CTD and MsiR (D133A) -CTD were first desalted into 20 mM phosphate buffer pH 8.0, 300 mM NaCl, 200mM KCl, 10% glycerol, 5mM beta-mercaptoethanol using HiTrap desaling column. The protein was then concentrated to 50uM, followed by dissolution of canavanine to 1mmol in desalting buffer, followed by titration of the protein with small molecules, at a volume of 0.4ul for each experiment, for a titration time of 0.8s, with a time interval of 120s for each titration. The affinity of MsiR-CTD, MsiR (D133A) -CTD and control buffer for small molecule canavanine was determined using an isothermal calorimeter. The analysis of the binding of small molecules to proteins by the software ORIGIN version 7.0 belongs to a single site binding model and common thermodynamic parameters were calculated (see table 2). From these results, it was found that the affinity of the C-terminal effector binding region of the MsiR mutein for binding to canavanine was increased by 1.5-fold.

TABLE 2 ITC measurement results of thermodynamic scanning

In the table: ka. Kd is the measured affinity, Δ G is the entropy change, Δ H is the enthalpy change, and Δ ST is the hydrophobic force.

The invention constructs a single-cell biosensor with coupled fluorescence signals and canavanine concentration based on the transcription regulatory factor MsiR mutant protein, detects the linear range of the single-cell biosensor, and has good linear relation between the fluorescence signal intensity and the canavanine concentration under the canavanine concentration of 0 mmol/L to 5.68 mmol/L. Compared with a wild type fluorescent value responding to canavanine, the transcriptional regulatory protein MsiR mutant protein disclosed by the invention has a two-fold increase, and the binding affinity of a C-terminal effector binding region of the MsiR mutant protein and the canavanine is increased by 1.5 times.

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.

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Claims

1. Rhizobium tianshanii transcriptional regulatory protein MsiR mutant protein, characterized in that: the mutant protein is a non-natural protein, and the mutant protein has the ability to specifically respond to concanavalinic acid, and the mutant protein is 1 of the wild-type MsiR The core amino acid related to the binding of canavanine is mutated: the 133rd position is mutated from aspartic acid (D) to alanine (A). The protein sequence of the mutant protein is shown in SEQ ID NO.1, and the nucleotide The sequence is as SEQ ID NO.2.

2. A polynucleotide, characterized in that: the polynucleotide encodes the mutein according to claim 1, comprising a polynucleotide encoding the full sequence of the mutein and the C-terminal of the mutein, wherein the full sequence of the mutein The protein sequence is shown in SEQ ID NO.1, the nucleotide sequence of the full sequence of the mutein is shown in SEQ ID NO.2; the nucleotide sequence of the C-terminal of the mutein is shown in SEQ ID NO.3, the mutein C The teloprotein sequence is shown in SEQ ID NO.4.

3. A vector, characterized in that: the vector contains the polynucleotide of claim 2.

4. A host cell, characterized in that: the host cell contains the vector of claim 3, or the polynucleotide of claim 2 is integrated into its genome.

5. A method for producing a mutein, characterized by: culturing the host cell of claim 4 under conditions suitable for expression, expressing the transcriptional regulatory protein MsiR mutein, and isolating the transcriptional regulatory protein MsiR mutein.

6 . The use of the mutein according to claim 1 , wherein the mutein is used for the construction of a biosensor responsive to canavanine or the detection of canavanine in vitro. 7 .

7. the purposes of mutein according to claim 6 is characterized in that: the detection of described in vitro canavanine comprises the following steps:

(1) pre-culturing Rhizobium tianshanii containing PTCV141 plasmid and PTM5 plasmid at a temperature of 28°C for more than 12 hours until entering a stable phase, wherein PTCV141 comprises the mutant protein of claim 1;

(2) the pre-cultivated Rhizobium Tianshan is inoculated in the fresh TY medium containing L-canavaline of different concentrations with the inoculum of 1:50;

(3) When the OD600 of the bacterial liquid reaches 0.6-0.8, the fluorescence intensity is measured after culturing for 48-72 hours.