High-salt-tolerance single-chain binding protein mutant and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a high-salt-tolerance single-chain binding protein mutant and application thereof.The amino acid sequence of the high-salt-tolerance mutant is shown as SEQ ID NO: 2 or SEQ ID NO: 3, compared with a wild type, a multi-site mutation / deletion SSB mutant SSBV1 contains V11I + A30C + I79F + T98S, SSBV2 contains V11I + A30C + I79F + T98S + 144-148del (GNQF), the binding efficiency of the SSBV1 and the SSBV2 under different salt concentrations is tested, and the high-salt-tolerance single-chain binding protein mutant can be obtained. Compared with wild escherichia coli SSB, the combination efficiency of the SSBV1 and the SSBV2 is remarkably improved under the conditions of different sodium chloride concentrations, different magnesium chloride concentrations and different ammonium sulfate concentrations, so that the application scene of the SSB is further expanded.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a highly salt-resistant single-chain binding protein mutant and application thereof. Background Art

[0002] Single-stranded DNA binding proteins (SSBs) are a class of proteins that can specifically bind to single-stranded DNA (ssDNA) and RNA. They function by forming nucleoprotein complexes with single-stranded nucleic acids in the system, while they show low preference for double-stranded DNA.

[0003] The main function of SSB is to participate in DNA replication and recombination in vivo, effectively keeping the melted DNA duplexes separated (preventing reannealing) and keeping the two strands in single-stranded form. Therefore, SSB can be used in all applications that require the preservation of DNA or RNA in single-stranded form, such as PCR, qPCR, RT-PCR, RT-qPCR, or other DNA or RNA modification enzyme reactions.

[0004] The most widely reported research is on SSB from Escherichia coli (E. coli, a protein of 178 amino acids), which can specifically bind to ssDNA to form a homotetramer and exert its effect. At the same time, the nucleic acid present in this complex can still serve as a substrate for DNA or RNA modifying enzymes without causing interference. The polymerase chain reaction (PCR) technology can be optimized by adding SSB, which stabilizes denatured DNA, prevents the formation of duplexes and protects ssDNA from digestion by nucleases. In sequencing reactions, when SSB is used together with a dedicated DNA polymerase for DNA sequencing reactions, it can increase signal intensity, reduce nonspecific signals and improve polymerization efficiency. Other applications include its use in combination with RecA in site-directed mutagenesis. At the same time, SSB can also promote the stability of RNA preservation, restriction endonuclease digestion reactions, the efficiency of reverse transcription in RT-PCR, and enhance the activity of T4DNA polymerase, among other types of applications.

[0005] Although SSB derived from E. coli has a wide range of functional applications in molecular biology, it cannot form a stable nucleoprotein complex at higher salt concentrations, showing sensitivity to high-salt environments and being unable to function in high-salt environments. This limits the type and amount of salt present in the reaction mixture of the formulation system (including samples), which in turn limits the scope of application of SSB (such as sequencing based on the Illumina platform). Therefore, protein engineering of SSB derived from E. coli so that it can still function in a system with high salt concentrations will help SSB expand its application scenarios in a range of applications in nucleic acid amplification and sequencing. Summary of the invention

[0006] In order to solve the problems mentioned in the background technology, the object of the present invention is to provide a highly salt-tolerant binding protein mutant.

[0007] The highly salt-tolerant binding protein mutant is SSBV1 or SSBV2.

[0008] The SSBV1 amino acid sequence is shown in SEQ ID NO:2, which is obtained by changing the 11th amino acid of the SSB amino acid sequence shown in SEQ ID NO:1 from V to I, the 30th amino acid from A to C, the 79th amino acid from I to F, and the 98th amino acid from T to S;

[0009] The SSBV2 amino acid sequence is shown in SEQ ID NO:3, which is obtained by changing the 11th amino acid of the SSB amino acid sequence shown in SEQ ID NO:1 from V to I, the 30th amino acid from A to C, the 79th amino acid from I to F, the 98th amino acid from T to S, and deleting the amino acids (GNQF) at positions 144 to 148.

[0010] The present invention also provides a nucleotide sequence encoding the highly salt-tolerant binding protein mutant. The nucleotide sequence of the highly salt-tolerant binding protein mutant is shown in SEQ ID NO:5 or SEQ ID NO:6.

[0011] The expression vector comprising the nucleotide sequence of the highly salt-tolerant binding protein mutant is also within the protection scope of the present invention.

[0012] The host cells obtained by the expression vector are also within the protection scope of the present invention.

[0013] The use of the highly salt-tolerant binding protein mutant, the expression vector or the host cell in nucleic acid amplification and sequencing also falls within the protection scope of the present invention.

[0014] The beneficial effects of the present invention are as follows: compared with the wild-type SSB, the multi-site mutation SSB mutant SSBV1 contains V11I+A30C+I79F+T98S, and the multi-site mutation and deletion SSB mutant SSBV2 contains V11I+A30C+I79F+T98S+144-148del(GNQF). The binding efficiency thereof under different salt concentrations was tested. Compared with the wild-type Escherichia coli SSB, under the conditions of different sodium chloride concentrations, different magnesium chloride concentrations and different ammonium sulfate concentrations, the binding efficiency of SSBV1 and SSBV2 was significantly improved, which is conducive to further expanding the application scenarios of SSB. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a comparison table of the relative fluorescence intensities of SSB, SSBV1 and SSBV2 in different concentrations of sodium chloride;

[0016] Figure 2 This is a comparison table of the relative fluorescence intensities of SSB, SSBV1 and SSBV2 in different concentrations of magnesium chloride;

[0017] Figure 3 This is a comparison table of the relative fluorescence intensities of SSB, SSBV1 and SSBV2 in different concentrations of ammonium sulfate. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the protection scope of the present invention.

[0019] Example 1: Construction of wild-type SSB and mutant strains

[0020] DNA fragments of SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 were synthesized respectively by a full sequence synthesis method, and the DNA fragments could transcribe and express wild-type and mutant single-stranded binding proteins with amino acid sequences of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The specific synthetic sequence included a purification tag 6×His sequence and a TAA termination codon added to the 3' end, and the synthetic fragments and the vector pET-28a were double-digested with restriction endonucleases XhoⅠ and BamHⅠ.

[0021] The enzyme digestion system (20 μL) is:

[0022] 10 μL of target gene fragment or plasmid, 2 μL of 10× Buffer, 1 μL of BamHI, 1 μL of XhoⅠ, and 20 μL of ddH2O; digest at 37℃ for 1 hour. Then, the enzymatic solution was identified by agarose gel electrophoresis. Then, the gel was cut to recover DNA.

[0023] After the target gene fragment was recovered, it was connected with T4 DNA ligase, the molar ratio of the vector to the target gene fragment was 1:10, and the connection system (25 μL) was: 10×T4 DNA Ligase Buffer 2.5 μL, DNA fragment 10 μL, vector DNA 4 μL, T4 DNA Ligase 1 μL, dH2O was made up to 25 μL; 18°C ​​overnight connection. The constructed recombinant expression vector pET-28a(+)-SSB / SSBV1 / SSBV2 was transformed into E.coli TOP10 competent cells, coated with LB agar plates with antibiotics, and cultured at 37°C overnight. Three positive transformants were extracted for double enzyme digestion identification, and the positive transformants with correct double enzyme digestion identification were sent to Shanghai Jierui Bioengineering Co., Ltd. for sequencing.

[0024] Pick the single positive colony that has been sequenced correctly and inoculate it into the competent cells of E. coli BL21 (DE3), spread it on the LA agar plate containing kanamycin, and culture it at 37°C overnight. Select the white colony and culture it at 37°C, 200r / min overnight. Under sterile conditions, mix the bacterial solution with 50% glycerol in a ratio of 1:1 to make the final concentration of glycerol reach 25%.

[0025] Example 2: Recombinant expression of wild-type SSB and its mutant proteins:

[0026] Streak the frozen bacterial solution corresponding to the strain to be expressed on the LB plate to activate the bacteria to ensure their growth during the fermentation process.

[0027] A single colony of the recombinant expression bacteria BL21 (DE3) / Pet28a-target gene was inoculated into a centrifuge tube containing 10 mL of LB liquid culture medium, and cultured in a shaking incubator at 37° C. and 200 rpm for 12-14 hours.

[0028] The bacterial solution was transferred to a flask containing 100 mL of LB liquid culture medium at a 1:100 inoculation rate and cultured in a shaker at 37°C and 200 rpm for 2 h until the OD600 reached approximately 0.6-0.8.

[0029] Add 0.1 M IPTG and control the final concentration of IPTG to 1 mM, and culture at 30°C and 200 rpm for 3 h.

[0030] Example 3: Bacteria disruption and protein purification:

[0031] Pour the fermentation broth into a 50ml centrifuge tube, centrifuge at 8000rpm for 25min and discard the supernatant. It can be inverted on absorbent paper. Then resuspend the bacteria with distilled water and centrifuge at 8000rpm for 15min and discard the supernatant. After washing twice, add Buffer A (50mM PBS, 500mM NaCl, pH7.4, 5mM imidazole) to resuspend the cells and pipette evenly.

[0032] During the ultrasonic cell disruption process, the bacteria should be placed on ice, and the ultrasonic cell crusher conditions are set as follows: 6mm amplitude, 60min, 50mL bacterial suspension, and ensure that the probe is close to but not touching the bottom.

[0033] Place the bacterial cell disruption solution in a precooled centrifuge, centrifuge at 4°C, 10000rpm for 30min, take the supernatant and store it at 4°C. The crude enzyme solution obtained by centrifugation may still contain some impurities. Usually, it is placed in a precooled centrifuge again before loading the Ni column, and centrifuged at 4°C, 10000rpm for 60min. For Ni column affinity chromatography, the filter disc used should be soaked in 1M NaOH in advance, ultrasonicated for 5h, and put into 20% ethanol after use. The solution used in the loading process must be filtered, and the lid should be sealed with tin foil after opening. Gradient elution should be used for the first loading. Use different concentrations of Buffer B (50mM PBS, 500mM NaCl, pH7.4, 25-300mM imidazole) to elute the impurities on the Ni column, and collect samples according to the UV peak diagram.

[0034] Example 4: Determination of protein concentration

[0035] Bradford protein quantification kit was used for determination, and the specific method is as follows:

[0036] (1) Take the protein standard BSA and dilute it with distilled water to a final concentration of 0.5 mg / mL;

[0037] (2) Add 2000, 1600, 1200, 800, 400, and 0 μL to centrifuge tubes respectively, add distilled water to make all centrifuge tubes reach 2000 μL, add 2000 μL Bradford staining solution to each centrifuge tube and mix well;

[0038] (3) Take 200 μL of the mixed solution and add it to ELISA plates in triplicate. Leave it at room temperature for 3-5 min.

[0039] (4) Turn on the microplate reader to measure the A595 value of the standard and obtain a standard curve;

[0040] (5) Take a new centrifuge tube, add 2000 μL of sample, and then add 2000 μL of Bradford solution and mix well;

[0041] (6) Take 200 μL of the mixture and add it to the ELISA plate in parallel, and place it at room temperature for 3-5 minutes;

[0042] (7) Turn on the microplate reader to measure the A595 of the sample and calculate the protein concentration in the sample based on the standard curve.

[0043] Example 5: Activity assay

[0044] Single-stranded DNA binding activity measurement

[0045] We used wild-type SSB from E. coli as a control and tested the ability of different SSB mutants to bind ssDNA in reaction buffer supplemented with different concentrations of sodium chloride, magnesium chloride, and ammonium sulfate. In this assay, we primarily detected changes in fluorescence of a fluorophore-labeled oligonucleotide (SEQ ID NO: 7). The oligonucleotide is structurally unique in that it forms a partial hairpin with a short 8-base pair (bp) double-stranded stem and a 24-base single-stranded loop. Both ends of the oligonucleotide are labeled with fluorescent dyes—FAM (carboxyfluorescein) and DABCYL (4-(4'-dimethylaminophenylazo)benzoic acid), the latter of which is used to quench FAM fluorescence. When the oligonucleotide is in its natural hairpin state, both the 5' and 3' ends of the stem are in close proximity, and FAM fluorescence is quenched by DABCYL. When SSB protein is added, it disrupts hydrogen bonds in the double-stranded stem and forces the oligonucleotide to dehybridize due to its ability to bind specifically to the nucleotide loop. Spatial separation of FAM and DABCYL allows measurement of dye fluorescence (Tang et al., Chemical Communications

[2011] ).

[0046] The test system is as follows:

[0047] 1) Add 0.125 μM molecular beacon to each reaction well;

[0048] 2) Then add 0.2 μM E. coli wild-type SSB and 0.2 μM mutant SSBV1-V2;

[0049] 3) Add a final volume of 50 μl reaction buffer 25 mM Tris-HCl (pH 7.4), 10 mM NaCl, and set up three parallel wells, and supplement the specified concentrations of NaCl and MgCl2 in the (NH4)2SO4 in the corresponding reaction wells. The supplemented concentrations are 0 mM to 1 M NaCl, 0 M to 300 mM MgCl2, and 0 M to 300 mM (NH4)2SO4;

[0050] 4) The reaction was then incubated at 37°C for 15 minutes and the fluorescence was measured on a qPCR instrument;

[0051] 5) All graphs were normalized to 100% of the initial response, measured in fluorescence units;

[0052] As attached Figure 1 As shown in Figures 2,3, with the increase of sodium chloride, magnesium chloride and ammonium sulfate concentrations, the two mutants showed improved binding effects compared to wild-type SSB.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A highly salt-tolerant binding protein mutant, characterized in that: The amino acid sequence of the highly salt-tolerant binding protein mutant is a sequence having more than 95% similarity with the SSB amino acid sequence shown in SEQ ID NO: 1 through amino acid substitution and deletion.

2. The highly salt-tolerant binding protein mutant according to claim 1, characterized in that The amino acid sequence of the highly salt-tolerant binding protein mutant is shown in SEQ ID NO: 2 or SEQ ID NO:

3.

3. The nucleotide sequence encoding the highly salt-tolerant binding protein mutant according to claim 2, characterized in that: The nucleotide sequence is shown in SEQ ID NO:5 or SEQ ID NO:

6.

4. An expression vector, characterized in that: A nucleotide sequence comprising the highly salt-tolerant binding protein mutant according to claim 3.

5. A host cell, characterized in that The method is obtained by transforming the expression vector described in claim 4 into cell engineering.

6. Use of the highly salt-tolerant binding protein mutant according to any one of claims 1 to 2, the expression vector according to claim 4 or the host cell according to claim 5 in nucleic acid amplification and sequencing.

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