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Mutant Taq enzyme and preparation method thereof

A mutant, nucleotide sequence technology, applied in the biological field, can solve problems such as limitations, achieve the effect of improving thermal stability and meeting the needs of industrial and research applications

Active Publication Date: 2014-01-15
GUANGDONG FAPON BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, with the development of medical science and technology, the requirements for polymerase performance are constantly increasing, and the original wild-type Taq DNA polymerase can no longer meet the requirements of special PCR technology. For example, the site-directed mutagenesis technology for functional genes requires polymerase to have high thermal stability etc.
Due to the structural defects of the original Taq DNA holoenzyme, it is limited in many application fields

Method used

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  • Mutant Taq enzyme and preparation method thereof
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  • Mutant Taq enzyme and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Site-directed mutagenesis of Taq enzyme plasmid

[0041] The plasmid containing the natural Taq enzyme gene was subjected to PCR amplification. The synthetic amplification primer is the mutation primer, which contains the site to be mutated and has replaced the base to be mutated. After amplification, the original master plate is digested with an enzyme, that is, the unmutated Taq gene template as the initial template is digested. The product of PCR, that is, the plasmid containing the mutated Taq enzyme gene was transformed into competent cells. The plasmid containing the mutant enzyme gene can be obtained by picking the clone.

[0042] 50μL PCR reaction system: 10×buffer (100mmol / L KCl, 100mmol / L (NH4) 2 SO 4, 200mmol / L Tris-HCl pH8.8, 20mmol / L MgSO 4 , 1% TritonX-100, 1mg / mL BSA) 5μL, 10mmol / L dNTP 0.5μL, 125ng each of mutation primers, 1U pfu enzyme, 1μL containing 50ng plasmid template, add ddH 2 0 to 50 μL. Reaction conditions: Denaturation at 95...

Embodiment 2

[0044] Example 2: Expression and purification of mutant modified Taq enzyme

[0045] Inoculate 50 μL strains in 5ml LB liquid medium and shake for 6-8h, then transfer to 250mL LB liquid medium, shake for 4-6h, add IPTG to a final concentration of 50mmol / L, and continue shaking overnight. Centrifuge 250mL of bacterial liquid in a 50mL centrifuge tube at 5000rpm for 10min to collect the bacterial cells, add 5mL of Binding buffer per 100mL of culture, add lysozyme to a final concentration of 1mg / mL, place on ice for 1h, shake at 4°C for 10min, add TritonX-100 to The final concentration is 1%, vigorously shake and mix, shake at 4°C for 10min, centrifuge at 5,000rpm for 30min, take the supernatant (reserved sample of 20μL bacterial cell crushing product), bathe in 75°C water for 1h, shake from time to time, centrifuge at 5000rpm for 30min, take the supernatant (Reserve 20 μL of crude enzyme solution). Gently mix the Ni-gel, take 1mL to the column, 1mL ddH 2 O washed twice, with 1...

Embodiment 3

[0046] Example 3 SDS-PAGE protein electrophoresis verification enzyme protein size and purity

[0047] The mutant Taq enzyme and the wild-type Taq enzyme obtained above were subjected to SDS-PAGE protein electrophoresis detection to identify the size and purity of the mutant Taq enzyme protein.

[0048] Method: Take 40ul of each sample, add 10ul of 5×Loading Buffer, bathe in boiling water for 10min, and load 5ul of sample. The constant pressure of the stacking gel is 100V, and the constant pressure of the separating gel is 180V. Coomassie brilliant blue staining.

[0049] Result: Please refer to figure 1 , wherein, 1 is the wild-type Taq enzyme, 2 and 3 are mutant Taq enzymes, and each electrophoresis band is pressed into a straight line in the stacking gel; it can be seen from the figure that the mutant Taq enzyme and the wild-type Taq enzyme band The size is consistent; the background of the rubber surface is clean, the sample bands are clear, and there are no visible ban...

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Abstract

The invention discloses a mutant Taq enzyme and a preparation method thereof. A modern genetic engineering technology is adopted, amino acids on a plurality of sites in an amino acid sequence of a natural Taq enzyme are mutated and recombined to obtain the mutant Taq enzyme, and specifically, a glutamic acid on the 626th site of the natural Taq enzyme is mutated into lysine, alanine on the 661st site of the natural Taq enzyme is mutated into a glutamic acid, isoleucine on the 665th site of the natural Taq enzyme is mutated into threonine, phenylalanine on the 667th site of the natural Taq enzyme is mutated into leucine, and isoleucine on the 707th site of the natural Taq enzyme is mutated into leucine. Compared with an unmodified wild-type Taq enzyme, the mutant Taq enzyme has the advantages that the activity and thermal stability of the enzyme are greatly improved by site-specific mutagenesis, and the requirements of industrial production and scientific research application can be better met.

Description

technical field [0001] The present application relates to the field of biotechnology, in particular to a mutant Taq enzyme and a preparation method thereof. Background technique [0002] In 1969, a Thermus aquaticus YT-1 was isolated from the volcanic hot springs of the Yellowstone National Forest Park in the United States. It grows in an environment rich in minerals at 70°C-75°C. In 1976, A. Chien isolated and purified the heat-resistant Taq DNA polymerase (referred to as Taq enzyme) from it. With the promotion and application of PCR technology, the research on the properties of Taq DNA polymerase is becoming more and more important. RandallK.Saiki applied it to PCR technology for the first time, making the PCR process realize automatic continuous cycle. [0003] Taq DNA polymerase is a thermostable DNA polymerase belonging to the DNA polymerase I family. The enzyme gene has a total length of 2496 bases, encodes 832 amino acids, and has a molecular weight of 94kD. It ha...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/63C12N1/21C12N15/10C12R1/19
CPCC12N9/1252C12Y207/07007
Inventor 范凌云李泓彦邓艳华
Owner GUANGDONG FAPON BIOTECH CO LTD
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