Method for extracting and purifying M-MLV reverse transcriptase

Abstract

The invention provides a method for extracting and purifying an M-MLV reverse transcriptase. The method comprises the steps of: activation and expression of engineering bacterium: activating M-MLV reverse transcriptase engineering bacteria, and adding IPTG for induced expression to obtain expressed engineering bacteria; treating the bacteria with lysozyme at room temperature, adding DTT and Twain-20, treating on ice for a period of time, and centrifuging to obtain a crude protein; slowly adding silica particles into the crude protein, stirring by a magnetic mixer, and centrifuging to remove sediment and obtain a supernatant; transferring the supernatant water to a water bath preheated to 60 DEG C, incubating, centrifuging, and removing precipitate to obtain the supernatant; passing the supernatant through a Ni-NTA column with Ni<2+> chelated, then desalinating to obtain purified M-MLV reverse transcriptase; and preserving the obtained M-MLV reverse transcriptase by adding glycerol to reach the volume fraction of 50% at -20 DEG. The invention has the advanategs of small enzyme activity loss, and high purity, high activity and high recovery amount of recovered protein.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for extracting and purifying M-MLV reverse transcriptase. Background technique [0002] Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT, M-MLV reverse transcriptase) is an RNA-dependent DNA polymerase whose gene source is the genome of Moloney Murine Leukemia Virus. It can use single-stranded RNA, DNA or RNA-DNA hybrids as templates for cDNA synthesis. [0003] M-MLV reverse transcriptase is obtained from prokaryotic expression bacteria (BL21(DE containing M-MLV Reverse Transcriptase gene) 3 ) engineering bacteria) for extraction and purification. [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 M-MLV reverse transcriptase uses low-temperature ultrasonic disruption, repeated ammonium sulfat...

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Problems solved by technology

[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 M-MLV reverse transcriptase uses low-temperature ultrasonic disruption, repeated ammonium sulfate precipitation, ion exchange, and chromatography. Purification with desalting, not only up to 50% of the protein will be lost in the early 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 the purified protease The activity is reduced, which affects the reverse transcription activity of the later enzyme

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