Improving the Activity of Phi29 DNA Polymerase by SaProt Prediction

Area of Science:

• Biochemistry
• Molecular Biology
• Enzyme Engineering

Background:

• Phi29 DNA polymerase possesses valuable strand displacement capabilities.
• Natural Phi29 DNA polymerase exhibits low catalytic efficiency, limiting its applications.

Purpose of the Study:

• To enhance the catalytic efficiency of Phi29 DNA polymerase.
• To explore the utility of the SaProt deep prediction model for enzyme modification.

Main Methods:

• 3D structure prediction using AlphaFold and validation with PROCHECK.
• Saturation mutagenesis calculations with SaProt to identify beneficial mutations.
• Site-directed mutagenesis to construct variants and assess enzyme activity.

Main Results:

• Identified 10 potential beneficial mutation sites using SaProt.
• Constructed 7 variants via site-directed mutagenesis.
• The A447R variant exhibited a 33-fold increase in activity compared to wild-type Phi29 DNA polymerase.

Conclusions:

• SaProt model shows significant potential for enzyme engineering.
• The engineered Phi29 DNA polymerase variant offers improved performance.
• This work provides a foundation for developing superior nucleic acid amplification tools.