Method for extracting purified Tth DNA polymerases

Abstract

The invention provides a method for extracting purified Tth DNA polymerases. The method comprises the following steps: taking engineering bacteria of Tth DNA polymerases for activation, and adding IPTG for inducible expression; adding DTT and tween-20 after the engineering bacteria are treated with lysozyme at room temperature, treating for a period of time on ice and then centrifuging to obtain crude protein; slowing adding silicon dioxide particles in the crude protein, stirring in a magnetic stirring apparatus, centrifuging to remove precipitates; allowing supernatant to flow through an Ni<2+>-chelated Ni-NTA column after the supernatant is supplemented with NaCl, then washing sequentially with washing buffer solutions which have 5 times of bed volume, and at last, eluting the target protein with an eluent, and receiving the eluent containing the target protein; desalting to obtain the purified Tth DNA polymerases; adding glycerol in the obtained purified Tth DNA polymerases, so that the volume fraction is 50%, and saving at -20 DEG C. The method is simple, small in enzyme activity loss, and high in purity and recovery capacity of the recovered protein.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for extracting and purifying Tth DNA polymerase. Background technique [0002] Tth DNA Polymerase (Tth DNA Polymerase) is a thermostable enzyme with a molecular weight of 94kDa. The source of the original enzyme is isolated from Thermus thermophilus HB-8. The enzyme is capable of DNA replication at 74°C. Tth DNA polymerase can catalyze the polymerization reaction in the 5'-3' direction of nucleotides in the presence of magnesium ions to form double-stranded DNA, and can also use RNA as a template along the 5'-3' direction in the presence of manganese ions. ' direction for nucleotide polymerization to occur. The enzyme also has 5'-3' exonuclease activity. Tth DNA polymerase can be used in PCR, reverse transcription, RT-PCR and primer extension reactions under higher temperature conditions. It is especially suitable for the development of one-step qRT-PCR kits. [0003] T...

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Problems solved by technology

[0003] The original source of Tth DNA polymerase was isolated from Thermus thermophilus (Thermus thermophilus) HB-8. Due to the expensive equipment needed for the cultivation of Thermus thermophilus, complex medium configuration, high culture temperature and long culture period , Errors are prone to occur during the cultivation process, resulting in low cultivation efficiency, and it is not easy to cultivate on a large scale. In addition, a long chromatography process is required in the purification process, resulting in a large decrease in enzyme activity, so the production capacity of the enzyme is extremely limited

[0004] At present, because the active protein is easily denatured and inactivated, the purification is required to be carried out under low temperature conditions (operated on ice). The conventional method of purifying Tth DNA polymerase uses low-temperature ultrasonic disruption, repeated ammonium sulfate precipitation, ion exchange, chromatography and desalination. Purification, not only up to 50% of the protein will be lost in the initial stage of purification, but also some genomic proteins are difficult to completely remove, and the presence of genomic proteins will lead to slow column loading, prolong the purification time, and reduce the protease activity obtained from purification , affecting the amplification and reverse transcription activity of the late enzyme

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