Expression of thermostable MMLV reverse transcriptase in Escherichia coli by directed mutation

Area of Science:

• Molecular Biology
• Enzyme Engineering

Background:

• Moloney murine leukemia virus reverse transcriptase (MMLV RT) is crucial for molecular biology techniques.
• Improving MMLV RT solubility and thermal stability enhances its utility.
• Directed mutagenesis and codon optimization are key strategies for enzyme improvement.

Purpose of the Study:

• To engineer a more functional MMLV RT with enhanced thermal stability and reduced ribonuclease H (RNase H) activity.
• To assess the impact of specific mutations on enzyme properties and performance.
• To develop a superior enzyme for applications like RT-PCR and RT-LAMP.

Main Methods:

• Site-directed mutagenesis was performed on the MMLV RT sequence, targeting the connection domain and RNase H active site.
• Two variants, rRT-L (without mutations) and rRT-K (with mutations), were expressed in E. coli.
• Enzyme purification, Western blotting, in silico analysis (IUpred 3.0), RT-PCR, and RT-LAMP assays were used for characterization.

Main Results:

• High expression levels of both rRT-L and rRT-K were achieved.
• In silico analysis indicated that mutations in rRT-K destabilized the RNase H domain.
• RT-PCR and RT-LAMP assays confirmed the RNase H inactivity of rRT-K.
• rRT-K demonstrated increased thermostability, tolerating up to 60°C in both assays.

Conclusions:

• Directed mutations and sequence optimization successfully enhanced MMLV RT thermostability and reduced RNase H activity.
• The engineered rRT-K enzyme shows promise for reducing non-specific products in one-step RT-PCR and RT-LAMP.
• This study provides a pathway for developing improved thermostable commercial reverse transcriptase enzymes.