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Engineering thermostable friend mouse leukemia virus reverse transcriptase through mutational combination.

Youhui Yang1, Zhong Li1, Jie Zhang1

  • 1School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China; Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, 300072, PR China.

Biochemical and Biophysical Research Communications
|March 31, 2025
PubMed
Summary

Engineered thermostable reverse transcriptase (RT) variants overcome heat limitations in molecular biology. The FrM5 variant shows enhanced stability and activity, improving research applications.

Keywords:
Friend murine leukemia virus (isolate FB29)Half-lifeMelting temperatureMutational combinationReverse transcriptase

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Reverse transcriptases (RTs) are crucial for molecular biology and medical research.
  • The lack of thermostability in conventional RTs limits their application.
  • Engineering thermostable RTs is essential for advancing research.

Purpose of the Study:

  • To engineer a thermostable reverse transcriptase (RT) derived from Friend mouse leukemia virus RT (FrMLV RT).
  • To characterize the enzymatic activity and stability of the engineered variant.
  • To investigate the mechanism underlying enhanced thermostability.

Main Methods:

  • Iterative rounds of mutational combination of FrMLV RT.
  • Rapid cell-free RT activity assays.
  • Thermostability assays including half-life determination and melting temperature analysis.
  • Template-primer (T/P) binding assays.

Main Results:

  • A thermostable variant, FrM5 (D178C/E280R/T284R/W291F/L581W), was successfully engineered.
  • FrM5 exhibited robust RT activity from 35-50 °C with T/P.
  • FrM5 demonstrated a half-life of ~20 min at 50 °C, significantly longer than the wild-type (<2 min).
  • FrM5 showed tighter binding to T/P, suggesting a protective mechanism against heat inactivation.

Conclusions:

  • The engineered FrM5 variant possesses significantly enhanced thermostability compared to wild-type FrMLV RT.
  • This thermostable RT is suitable for a broader temperature range, improving molecular biology applications.
  • Tighter binding to template-primer is a key factor in the enhanced heat resistance of FrM5.