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Mutant TaqDNA polymerase and purification method thereof

A polymerase and mutant technology, applied in the biological field, can solve the problems of increasing cost, increasing the content of miscellaneous proteins, increasing the complexity of the process, etc., and achieves the effect of strong anti-inhibitory effect and high thermal stability.

Active Publication Date: 2018-07-10
天津强微特生物科技有限公司
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Problems solved by technology

Most of the subsequent articles on the purification method of Taq DNA polymerase have also adopted the ion exchange process. Although the ion exchange process can increase the loading capacity, it will lead to the content of foreign proteins due to its non-specific elution. will increase, and if a certain purity requirement is met, other steps of purification are required, which will increase the complexity of the process and increase the cost

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  • Mutant TaqDNA polymerase and purification method thereof
  • Mutant TaqDNA polymerase and purification method thereof
  • Mutant TaqDNA polymerase and purification method thereof

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Embodiment Construction

[0021] The present invention uses molecular biology technology to remove the 5'-3' exonuclease activity of natural Taq DNA polymerase, and mutate amino acids at multiple sites, and then recombine into the expression vector after confirming the mutant type by sequencing. The mutant Taq DNA polymerase has higher thermal stability than the wild-type Taq DNA polymerase, and has a stronger anti-inhibition effect on impure substances in biological samples such as blood or feces. The mutant Taq DNA polymerase can meet the increasing needs of scientific research, clinical and industrial applications.

[0022] In the present invention, the above-mentioned mutations specifically relate to the C-terminal 281-amino acid of the wild-type Taq DNA polymerase, and E507R, E742A and A743H mutations are introduced into the amino acid sequence of the natural Taq DNA polymerase. In one embodiment of the present invention, the amino acid sequence of its mutant Taq DNA polymerase is as shown in SEQ ...

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Abstract

The invention discloses a mutant TaqDNA polymerase and a purification method thereof in escherichia coli. By adoption of a modern gene engineering technology, a natural TaqDNA polymerase is mutated toremove 5'-3' DNA exonuclease activity, and E507R, E742A and A743H mutants are introduced, wherein amino acid positions are determined according to a wild type TaqDNA polymerase. Compared with the wild type Taq DNA polymerase, the mutant Taq DNA polymerase has advantages of high thermal stability and resistance to inhibiting effects caused by impure components in biological samples to a polymerase, thereby meeting scientific research and clinical application requirements. In addition, in a purification process, by adoption of a specific affinity column which is simple, convenient, quick and high in yield, the obtained mutant TaqDNA polymerase is high in quality, low in cost and quite suitable for industrial large-scale production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mutant Taq DNA polymerase and a purification method thereof. Background technique [0002] Taq DNA Polymerase was isolated from a Thermus aquaticus YTI strain by A. Chien. The yT1 strain is a kind of eubacterium melophilus that was isolated from the volcanic hot springs of the Yellowstone National Forest Park in the United States in 1969, and is suitable for growing at 70-75°C. The full length of Taq DNA polymerase gene is 2496 bases, encoding 832 amino acids, the enzyme protein molecule is 94 KDa, each enzyme molecule can extend about 150 nucleotides per second at 80 °C, and the elongation rate is greater than 60 at 70 °C nucleotides / sec. The enzyme activity of TaqDNA polymerase can last for 40 minutes at 95°C, and about 50% of the enzyme activity can be maintained by heating at 97.5°C for 5-6 minutes. The thermostability of Taq DNA polymerase is the prerequisite for the automa...

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12N15/10C12R1/19
CPCC12N9/1252C12N15/70C12N2800/101C12P19/34C12Q1/686C12Y207/07007C12Q2521/101
Inventor 王亮王磊郑春阳
Owner 天津强微特生物科技有限公司
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