Highly salt-tolerant single-chain binding protein mutant and its application
By performing multiple-point mutations and deletions on Escherichia coli SSB, highly salt-tolerant binding protein mutants SSBV1 and SSBV2 were prepared, which solved the problem of low binding efficiency of SSB in high-salt environments and achieved efficient application under high-salt conditions.
Patent Information
- Application Number
- CN202411498365.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-25
- Publication Date
- 2025-09-30
- Estimated Expiration
- 2044-10-25
AI Technical Summary
The single-stranded DNA binding protein (SSB) from Escherichia coli cannot form a stable nucleoprotein complex in a high-salt environment, which limits its application in high-salt environments.
By protein engineering of Escherichia coli SSB, highly salt-tolerant binding protein mutants SSBV1 and SSBV2 were prepared. Specifically, multiple point mutations and deletions were performed on the amino acid sequence to form SSBV1 (V11I+A30C+I79F+T98S) and SSBV2 (V11I+A30C+I79F+T98S+144-148del(GNQF)), so as to improve their binding efficiency in high-salt environments.
The mutants SSBV1 and SSBV2 significantly improved their binding efficiency at different salt concentrations, expanding their application scenarios in nucleic acid amplification and sequencing.
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Figure CN119192309B_ABST
Abstract
Description
Technical Field
[0001] The present 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, and they show low preference for double-stranded DNA.
[0003] SSBs primarily participate in DNA replication and recombination in vivo, effectively maintaining the separation of melted DNA duplexes (preventing reannealing) and maintaining both strands in single-stranded form. Therefore, SSBs are useful in all applications requiring the preservation of DNA or RNA in single-stranded form, such as PCR, qPCR, RT-PCR, RT-qPCR, or reactions involving other DNA- or RNA-modifying enzymes.
[0004] The most widely studied protein is SSB (a 178-amino acid protein) from Escherichia coli. It specifically binds to ssDNA to form a homotetramer, allowing the nucleic acid present in this complex to serve as a substrate for DNA- or RNA-modifying enzymes without interfering. Polymerase chain reaction (PCR) technology can be optimized by adding SSB. SSB stabilizes denatured DNA, preventing duplex formation and protecting ssDNA from nuclease digestion. In sequencing reactions, when used with specialized DNA polymerases for DNA sequencing reactions, SSB can increase signal intensity, reduce nonspecific signals, and improve polymerization efficiency. Other applications include its combination with RecA in site-directed mutagenesis. SSB can also promote RNA storage stability, digestion with restriction endonucleases, enhance the efficiency of reverse transcription in RT-PCR, and enhance the activity of T4 DNA polymerase, among other applications.
[0005] Although E. coli-derived SSB 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 (including samples) of the formulation system, which in turn limits the scope of application of SSB (such as sequencing based on the Illumina platform). Therefore, protein engineering of E. coli-derived SSB so that it can still function in a system with high salt concentrations will help expand the application scenarios of SSB in a series of applications of nucleic acid amplification and sequencing. Summary of the Invention
[0006] In order to solve the problems mentioned in the background technology, the purpose 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 144 to 148 (GNQF).
[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). Their binding efficiency under different salt concentrations was tested. Compared with the wild-type Escherichia coli SSB, under 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 clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention are within the scope of protection 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 can transcribe and express wild-type and mutant single-chain binding proteins with amino acid sequences of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The specific synthetic sequence includes a purification tag 6×His sequence and a TAA termination codon added to the 3' end. The synthetic fragments and the vector pET-28a were double-enzymed with restriction endonucleases XhoⅠ and BamHI.
[0021] The enzyme digestion system (20 μL) is:
[0022] Prepare 10 μL of target gene fragment or plasmid, 2 μL of 10× Buffer, 1 μL of BamHI, 1 μL of XhoⅠ, and ddH2O to a total of 20 μL. Digest the fragment at 37°C for 1 hour. Identify the digest by agarose gel electrophoresis. Then, excise the gel and recover the DNA.
[0023] After recovering the target gene fragment, ligation was performed using T4 DNA ligase at a molar ratio of vector to target gene fragment of 1:10. The ligation system (25 μL) consisted of 2.5 μL of 10× T4 DNA Ligase Buffer, 10 μL of DNA fragment, 4 μL of vector DNA, 1 μL of T4 DNA Ligase, and dH2O to a final volume of 25 μL. Ligation was performed overnight at 18°C. The constructed recombinant expression vector pET-28a(+)-SSB / SSBV1 / SSBV2 was transformed into E. coli TOP10 competent cells, plated on antibiotic-containing LB agar plates, and cultured overnight at 37°C. Three positive transformants were extracted and identified by double enzyme digestion. Positive transformants identified by double enzyme digestion were sent to Shanghai Jierui Biotechnology Co., Ltd. for sequencing.
[0024] A single positive colony that has been sequenced correctly was inoculated into competent E. coli BL21(DE3) cells, plated onto LA agar plates containing kanamycin, and cultured overnight at 37°C. White colonies were selected and cultured overnight at 37°C, 200 rpm. Under sterile conditions, the bacterial suspension was mixed with 50% glycerol at a 1:1 ratio, for a final glycerol concentration of 25%.
[0025] Example 2: Recombinant expression of wild-type SSB and its mutant proteins:
[0026] Streak the frozen bacterial liquid corresponding to the strain to be expressed on an LB plate to activate the bacteria to ensure 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 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 medium at a 1:100 inoculation ratio and cultured in a shaking incubator 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. The tube can be inverted on absorbent paper. Then, resuspend the bacteria in 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. The ultrasonic cell disruptor 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 cell suspension in a pre-cooled centrifuge and centrifuge at 4°C, 10,000 rpm, for 30 minutes. The supernatant should be stored at 4°C. The crude enzyme solution obtained from the centrifugation may still contain some impurities and should usually be placed in a pre-cooled centrifuge again and centrifuged at 4°C, 10,000 rpm, for 60 minutes before loading onto the Ni column. For Ni column affinity chromatography, the filter disc to be used should be soaked in 1M NaOH in advance, sonicated for 5 hours, and then placed in 20% ethanol after use. The solution used during the loading process must be filtered, and the lid should be sealed with aluminum foil after opening. Gradient elution should be used for the first loading. Elute the impurities from the Ni column using various concentrations of Buffer B (50mM PBS, 500mM NaCl, pH 7.4, 25-300mM imidazole). Collect samples based on the UV peak profile.
[0034] Example 4: Determination of protein concentration
[0035] The Bradford protein quantification kit was used for determination. 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 the centrifuge tubes, add distilled water to make all the 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 the ELISA plate, set up three parallel plates, and place it at room temperature for 3-5 minutes.
[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) Add 200 μL of the mixture to three parallel plates and place them 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] Using wild-type Escherichia coli SSB as a control, we tested the ability of different SSB mutants to bind ssDNA in a reaction buffer supplemented with varying concentrations of sodium chloride, magnesium chloride, and ammonium sulfate. In this assay, we primarily detected changes in the fluorescence of a fluorophore-labeled oligonucleotide (SEQ ID NO: 7). This oligonucleotide's structural peculiarity allows it to form a partial hairpin with a short, 8-base-pair (bp) double-stranded stem and a 24-base single-stranded loop. It is also labeled at both ends with the fluorescent dyes FAM (carboxyfluorescein) and DABCYL (4-(4'-dimethylaminophenylazo)benzoic acid), the latter of which quenches FAM fluorescence. When the oligonucleotide is in its native hairpin state, the 5' and 3' ends of the stem are in close proximity, and FAM fluorescence is quenched by DABCYL. When SSB protein is added, its specific binding to the nucleotide loop disrupts hydrogen bonds in the double-stranded stem and forces the oligonucleotide to dehybridize. Spatial separation of FAM and DABCYL allows for 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, 0.2 μM E. coli wild-type SSB and 0.2 μM mutant SSBV1-V2 were added;
[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. Supplement the specified concentrations of NaCl and MgCl2 in the corresponding reaction wells with (NH4)2SO4. The supplemented concentrations range from 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 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 increasing concentrations of sodium chloride, magnesium chloride, and ammonium sulfate, 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 scope of protection 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 shown in SEQ ID NO: 2 or SEQ ID NO:
3.
2. The nucleotide sequence encoding the highly salt-tolerant binding protein mutant according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO: 5 or SEQ ID NO:
6.
3. An expression vector, characterized in that Comprising the nucleotide sequence of the highly salt-tolerant binding protein mutant according to claim 2.
4. A host cell, characterized in that The method is obtained by transforming the expression vector described in claim 3 into an engineered cell.
5. Use of the highly salt-tolerant binding protein mutant according to claim 1, the expression vector according to claim 3 or the host cell according to claim 4 in nucleic acid amplification and sequencing.