A novel thermostable RNase HI sequence mutant, its preparation method, and its application.

CN121801868BActive Publication Date: 2026-06-30BEIJING TRANSGEN BIOTECH CO LTD +1
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
BEIJING TRANSGEN BIOTECH CO LTD
Filing Date
2026-01-15
Publication Date
2026-06-30

AI Technical Summary

Technical Problem

Existing thermostable RNase HI has low heterologous expression levels and high production costs, making it difficult to meet the needs of molecular biology applications under high-temperature reaction conditions.

Method used

A novel sequence mutant of thermostable RNase HI was designed by fusing a new sequence to the carboxyl terminus of wild-type thermostable RNase HI. The mutant was then overexpressed and purified using the recombinant expression plasmid pET-21a-RNaseHIMut1, including PEI precipitation, Ni affinity chromatography, and ion exchange chromatography, to enhance enzyme activity and thermostable properties.

Benefits of technology

It significantly improved the heterologous expression level and enzyme activity of thermostable RNase HI, reduced production costs, and is suitable for molecular biology applications under high-temperature reaction conditions.

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Abstract

This invention discloses a novel thermostable RNase HI sequence mutant, its preparation method, and its applications. First, it discloses the thermostable RNase HI sequence mutant with the amino acid sequence shown in SEQ ID NO.1. It further discloses the preparation method and applications of the aforementioned thermostable RNase HI sequence mutant. This invention's thermostable RNase HI sequence mutant incorporates a novel sequence fused to the carboxyl terminus of wild-type thermostable RNase HI. It can be overexpressed in prokaryotic hosts and purified in large quantities via affinity chromatography and ion exchange chromatography, significantly increasing yield. Under the same conditions, it exhibits significantly improved enzyme activity and thermostable properties compared to the wild type, which is of great significance for optimizing enzyme performance and greatly reduces the production cost of commercially available thermostable RNase HI. This will promote the widespread application of thermostable RNase HI in various fields.
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