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Terminal deoxyribonucleoside transferase variant and application thereof

A deoxyribonucleoside and transferase technology, applied in the field of terminal deoxyribonucleoside transferase variants, can solve the problems of low DNA polymerase activity and difficult enzymatic oligonucleotides

Active Publication Date: 2019-10-15
天津中合基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the activity of existing DNA polymerases is low, and how to achieve efficient and controllable enzymatic oligonucleotides is still a big problem

Method used

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  • Terminal deoxyribonucleoside transferase variant and application thereof
  • Terminal deoxyribonucleoside transferase variant and application thereof
  • Terminal deoxyribonucleoside transferase variant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Obtaining of TDT protein

[0038] 1. Amino acid sequence of TDT protein

[0039] The wild-type TDT amino acid sequence is SEQ ID NO: 1;

[0040] 2. Construction of expression vector

[0041] All gene sequences that can synthesize the amino acid sequence shown in SEQ ID NO: 1 can be used for the construction of expression vectors, and the gene sequences are constructed into expression vector pET-28a (Novagen, Kan+, see figure 1 )Restriction sites Nde I and xho Between I, obtain recombinant plasmid, named as pET-28a-TDT. pET-28a is described in the examples, but the expression vector of the present invention is not limited thereto.

[0042] 3. gene expression

[0043] In order to detect the activity of TDT enzyme in vitro, the enzyme was expressed and purified exogenously in Escherichia coli. The host bacterium described in the embodiment is E. coli BL21 (DE3).

[0044] (1) Transfer the Escherichia coli expression recombinant plasmid pET-28a-TDT ...

Embodiment 2

[0057] Example 2 Obtaining of Mutants

[0058] Through computer simulation, the substrate molecules and proteins were docked, the catalytic mechanism was analyzed, the D396E and K403M variants were obtained, and the target variants were introduced by PCR. The expression and purification conditions of the variants are the same as those of the wild-type TDT expression and purification conditions in Example 1.

Embodiment 3

[0059] Example 3 Function Verification

[0060] The nucleotide used in this embodiment is a nucleotide with a phosphate added to the 3'-OH end as a blocking group, and the 5' end has three phosphates.

[0061] 1 . In vitro pure enzyme reaction, the reaction schematic diagram see image 3 .

[0062] 2. TDT reaction system: 100 mM NaCl, 0.25 mM CoCl 2 , 50 mM KAc, 10 mM Mg(Ac) 2 , pH6.8. Substrate: 100 µM oligo (14 bp), 1 µM 3'-modified nucleotides. Enzyme: 0.1 mM TDT wild type and mutants.

[0063] Reaction conditions: Incubate at 35°C for 1 min and immediately add 0.5 M EDTA to terminate the reaction, inactivate at 70°C (to denature the protein and release the oligo chain) and centrifuge to take the supernatant for urea-denatured PAGE gel.

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Abstract

The invention provides a terminal deoxyribonucleoside transferase variant and application thereof. The coupling efficiency of a nucleotide modified with a 3'-OH terminal is improved by introducing a mutation at a specific site in a wild type amino acid sequence, and nucleic acid molecules are synthesized by enzymatic reaction efficiently and controllably. The invention further provides a method for synthesizing the nucleic acid molecules without a template strand.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a terminal deoxyribonucleoside transferase variant and its application. Background technique [0002] DNA is the carrier of life information, and obtaining DNA is the first step in researching, transforming and creating life. DNA synthesis technology is the most important common basic support technology in the field of life sciences. There are two main methods for the de novo synthesis of oligonucleotides: chemical synthesis (solid-phase phosphoramidite triester synthesis) and biosynthesis (enzymatic synthesis). Since the 1950s, chemical methods and enzymatic methods have developed separately, and the core key to oligonucleotide synthesis has become controllable, efficient, and continuous synthesis. In 1981, the solid-phase phosphoramidite synthesis method was invented, which uses porous glass (controlled pore glass, CPG) or polystyrene (polystyrene, PS) as a solid phase carrie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12P19/34
CPCC12N9/1264C12P19/34C12Y207/07031
Inventor 江会锋彭凯逯晓云阮江星王千卢丽娜
Owner 天津中合基因科技有限公司
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