A method of preparing high-performance M-MLV reverse transcriptase

Abstract

A method of preparing high-performance M-MLV reverse transcriptase is disclosed. The method includes a step of performing mutant cloning construction, with plasmid sequences of constructed mutants being sequenced for confirmation to obtain seventeen mutant plasmids; a step of mutant screening, namely a step of transforming wild-type pET28b-M-MLV and the seventeen mutant plasmids into escherichia coli competent cells, culturing, inducing mutant enzyme expression, collecting bacterium cells to obtain a mutant enzyme coarse extract liquid, and subjecting mutant enzyme coarse extract liquid having protein expression to reverse transcription activity screening at different temperatures; and a step of culturing the mutants positive in activity screening in a liquid medium, inducing protein expression, collecting bacterium cells, and performing purification to obtain the M-MLV reverse transcriptase. A combination of molecular rational design and function screening is adopted by the method, the M-MLV reverse transcriptase having high thermal stability can be obtained only by a small range of screening, and the thermal stability is that the M-MLV reverse transcriptase can resist a temperature of 65 DEG C.

Description

technical field [0001] The invention relates to a method for preparing a biocatalyst, in particular to a method for preparing a high-performance M-MLV reverse transcriptase. Background technique [0002] Modern biomedicine is based on the "central dogma" of molecular biology. Studying gene function through gene expression is one of the core issues in modern biomedicine. Since RNA, the product of gene expression, is easily degraded, most studies on gene expression require the reverse transcription of RNA into cDNA as the research object. Especially in recent years, with the rapid development of high-throughput sequencing technology, RNA sequencing has been widely used in the frontier research of biomedicine. Many aspects such as animal and plant genetics and breeding have immeasurable potential. RNA sequencing also needs to first convert the template RNA into cDNA to construct a sequencing library. In these processes, a very important biocatalyst - reverse transcriptase i...

AI Technical Summary

Problems solved by technology

The disadvantages are: because mutations are randomly introduced, there are too many invalid mutations or inactivating mutations, and the workload of later screening is large and the hit rate is low.

The compartmentalized ribosome display (CRD) evolution in vitrotechnique used for screening needs to use a cell-free expression system, which is expensive

The heat resistance of the highest thermostable M-MLV reverse transcriptase reported so far is 60°C, and the heat resistance is relatively low

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