Stabilization and activation of recombinant human immunodeficiency virus-1 reverse transcriptase-P66

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Human immunodeficiency virus-1 reverse transcriptase-p66 (HIV-1 RT) is crucial for viral replication.
• HIV-1 RT exhibits instability at refrigeration temperatures (4°C) in standard buffers.
• Enzyme stability and activity are critical for research and therapeutic applications.

Purpose of the Study:

• To investigate methods for improving the solution stability of HIV-1 RT-p66.
• To explore factors influencing the activity and stability of HIV-1 RT-p66.
• To elucidate the potential mechanisms behind observed enzyme activity changes.

Main Methods:

• Assessing HIV-1 RT-p66 stability in a standard buffer at 4°C and -70°C.
• Evaluating the effect of (rA)n(dT)12-18 template-primer on enzyme stability.
• Investigating the impact of 1.0 M ammonium phosphate on enzyme activity and stability.
• Analyzing enzyme reactivation and the influence of chaotropic and antichaotropic salts.

Main Results:

• HIV-1 RT-p66 is unstable at 4°C but stable when frozen at -70°C.
• Incorporation of (rA)n(dT)12-18 template-primer significantly enhanced the solution stability of dilute enzyme.
• 1.0 M ammonium phosphate induced a reproducible ~260% activation of enzyme activity.
• Inactivated enzyme could be reactivated to the same elevated level, indicating reversible interconversion.

Conclusions:

• Template-primer incorporation is a viable strategy to improve HIV-1 RT-p66 solution stability.
• Ammonium phosphate acts as a potent activator for HIV-1 RT-p66.
• The reversible activation suggests interconversion between monomeric and dimeric forms of p66, influenced by salt conditions.