Expression of Codon-Optimized Gene Encoding Murine Moloney Leukemia Virus Reverse Transcriptase in Escherichia coli

Area of Science:

• Molecular Biology
• Enzyme Engineering
• Biotechnology

Background:

• Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is crucial for cDNA synthesis and widely used in reverse transcription polymerase chain reaction (RT-PCR).
• RT-PCR is a key method for detecting SARS-CoV-2, the virus responsible for the COVID-19 pandemic.

Purpose of the Study:

• To enhance the production yield and enzymatic performance of MMLV-RT.
• To optimize MMLV-RT expression in an E. coli system through codon and culture condition adjustments.

Main Methods:

• Codon optimization and culture condition refinement were employed in an E. coli expression system.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were used to assess enzyme overexpression.
• Nickel affinity chromatography was utilized for single-step purification of the recombinant enzyme.

Main Results:

• Successful overexpression of MMLV-RT was achieved, with a significant 85-fold increase in yield (from 0.002 g L⁻¹ to 0.175 g L⁻¹).
• Purified MMLV-RT demonstrated promising qualitative and quantitative reverse transcriptase activity.
• The optimized enzyme showed potential for use in RT-PCR assays.

Conclusions:

• The study presents effective strategies for improving recombinant MMLV-RT production and performance.
• Enhanced MMLV-RT offers a valuable tool for molecular biology applications, including sensitive pathogen detection.