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Novel fusion protein of mispairing binding protein and cellulose binding domain 3 and method thereof for removing errors in DNA (Desoxvribose Nucleic Acid) synthesis at high flux

A fusion protein and binding protein technology, applied in the field of high-throughput correction of DNA synthesis errors, can solve problems such as DNA synthesis errors, poor quality, complex oligonucleotide library components, etc., to achieve cost reduction, low price, high error With the effect of removing ability

Active Publication Date: 2014-04-30
UNIV OF SCI & TECH OF CHINA
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  • Description
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AI Technical Summary

Problems solved by technology

2) The composition of the oligonucleotide library synthesized by the chip is very complex
After the synthetic oligonucleotides are cut from the chip, a library containing thousands of different types of oligonucleotides will be formed, and when these oligonucleotides are used for subsequent full-length gene assembly, due to the The diversity of sequences will bring great difficulties to the assembly of full-length genes
3) The quality of oligonucleotides synthesized by the chip is worse than those synthesized by traditional methods
However, whether MutS can be used for high-throughput error correction of chip-synthesized DNA has not been systematically studied.
Although MutS from Thermophytes and MutS from Escherichia coli have been applied to DNA error correction, and MutS from Thermophytes has also been combined with MBP and then passed through using a substrate that can bind to MBP. MutS bound to mismatched DNA was removed by centrifugation[23], but MutS derived from Escherichia coli has not been studied in this regard, and none of them immobilized MutS on cellulose to form specific binding to mismatched bases The study of the column of DNA, and neither combined with the CBM domain to fix the MutS protein to amorphous cellulose (amorphous cellulose) and then formed a MutS cellulose column (MCSC) to correct DNA synthesis errors, while other Amorphous cellulose is much less costly than gel media
And there are no studies applying this technique to the study of high-throughput error correction of synthesized DNA in chips

Method used

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  • Novel fusion protein of mispairing binding protein and cellulose binding domain 3 and method thereof for removing errors in DNA (Desoxvribose Nucleic Acid) synthesis at high flux
  • Novel fusion protein of mispairing binding protein and cellulose binding domain 3 and method thereof for removing errors in DNA (Desoxvribose Nucleic Acid) synthesis at high flux
  • Novel fusion protein of mispairing binding protein and cellulose binding domain 3 and method thereof for removing errors in DNA (Desoxvribose Nucleic Acid) synthesis at high flux

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0040] Preparation of amorphous cellulose:

[0041] 0.2g of microcrystalline cellulose and 0.6mL of water were thoroughly mixed in a 50mL centrifuge tube to prepare a cellulose suspension, and then 10mL of ice-cold concentrated phosphoric acid (>85%) was added. Add slowly while mixing vigorously. This cellulose mixture will quickly become transparent. Place on ice for 1 h with occasional stirring. Then 40 mL of ice-cold water was added in four portions. Stir vigorously when adding to avoid lumps of cellulose. The precipitated cellulose was centrifuged at 5000 x g for 20 min at 4°C. Discard the supernatant, resuspend the pellet in ice water, centrifuge again, and discard the supernatant, repeat 4 times to remove phosphoric acid. Add 1mL2MNa 2 CO 3 Add 40mL of ice-cold water to the cellulose suspension to neutralize residual phosphoric acid, centrifuge to remove the supernatant, and wash with water twice. If the pH of the cellulose is 5-7, the preparation of amorphous cel...

Embodiment 1

[0051] The preparation of embodiment 1, eMutS and tMutS protein

[0052] 1. Construction of eMutS and tMutS protein expression vectors

[0053] 1). Acquisition of EcoMutS gene:

[0054] Constructed with the plasmid pET-32a-eMutS (inserting the EcoMutS gene (GenBank: HG738867.1) into the EcoRI and HindIII sites of pET-32a, see Bi Lijun et al., AnthonyE.G.Cass (2002) DNA mismatch repair gene High-efficiency expression of mutS and identification of expression product activity. Acta Bioengineering 18(5):536-540) as a template, design specific amplification primers, and use PrimeSTAR HS DNA polymerase (Bao Biological Engineering (Dalian) Co., Ltd.) for PCR , the obtained product fragment is an EcoMutS-NS fragment containing the EcoMutS gene ("-NS" indicates that the fragment contains restriction sites N: NheI and S: SacI, the following are similar).

[0055] PCR system of EcoMutS-NS gene:

[0056]

[0057]

[0058] PCR program

[0059]

[0060] 2). Obtaining of TaqMutS...

Embodiment 2

[0140] Embodiment 2: the construction of MCSC:

[0141] The MutS protein fused with CBM3 can be immobilized on cellulose by utilizing the stable and specific binding ability between CBM3 and amorphous cellulose, and then the cellulose immobilized with MutS protein can be packed into an empty column to form MutS cellulose column (MCSC).

[0142] The specific operation steps are:

[0143]In order to assemble a standard 2cm-high MCSC, 1ml of cellulose (20mg / ml) was mixed with 1200pmol of MutS fusion protein, combined at room temperature for 10 minutes, and the cellulose bound to MutS was loaded into an empty column (0.4cm×7cm, Sangon Bioengineering (Shanghai) Co., Ltd.) formed a column with a height of 2 cm and a diameter of 0.4 cm. After the column is stabilized, add 1 ml of binding buffer [5 mM MgCl2, 100 mM KCl, 20 mM Tris-HCl (pH7.6), 1 mM DTT] to wash away unbound MutS protein. According to the above-mentioned MCSC packing method, a total of five MCSCs ( figure 1 ), the ...

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Abstract

The invention discloses a novel fusion protein of a mispairing binding protein and a cellulose binding domain 3, establishes a method for rapidly immobilizing the fusion protein on cellulose, and a method for constructing a mispairing binding protein cellulose chromatographic column. The invention further establishes a method for removing errors in oligonucleotides synthesized by a chip at high flux by using the mispairing binding protein cellulose chromatographic column. Meanwhile, the method disclosed by the invention is capable of correcting the errors in the DNA synthesis at high flux.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to the field of constructing a fusion protein MutS capable of removing synthesis errors in oligonucleotides and DNA and its application. The invention relates to a method for immobilizing and constructing a cellulose column by using the immobilized recombinant mismatch binding protein MutS. The present invention also relates to a method for correcting synthesis errors of chip-synthesized oligonucleotides and their assembled DNA using a MutS cellulose chromatographic column, especially using this chromatographic column for immobilizing recombinant proteins to correct DNA efficiently, quickly, and with high throughput The wrong way to synthesize. Background technique [0002] Gene de novo synthesis technology is playing an increasingly important role in synthetic biology, systems biology and general life science research [1-6]. The commonly used strategy for de no...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/10B01J20/26B01D15/08
Inventor 洪泂高晓连宛雯周小川
Owner UNIV OF SCI & TECH OF CHINA
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