Serine integrase chimeras with activity in E. coli and HeLa cells

Area of Science:

• Molecular Biology
• Genomic Engineering
• Enzymology

Background:

• Large serine recombinases are increasingly used for genomic engineering due to their factor-independence and unidirectionality.
• Applications include site-directed vector integration and cassette exchange across various organisms.
• Understanding and modifying serine integrase specificity and efficiency is crucial for advancing these applications.

Purpose of the Study:

• To explore a hybridization strategy for altering serine integrase specificity and improving efficiency.
• To create and characterize chimeric serine integrases derived from phiC31, phiBT1, and TG1 integrases.
• To investigate the activity and substrate specificity of these novel hybrid enzymes.

Main Methods:

• Construction of chimeric serine integrases by combining amino- and carboxy-terminal portions of phiC31, phiBT1, and TG1 integrases.
• Testing the activity of generated hybrid integrases (phiBT1-phiC31 (BC) and phiC31-TG1 (CT)) in E. coli.
• Evaluation of hybrid integrase activity on native and hybrid att-sites.
• Assessment of a specific BC hybrid's activity in human HeLa cells.

Main Results:

• Several phiBT1-phiC31 (BC) and phiC31-TG1 (CT) hybrid integrases demonstrated activity in E. coli.
• BC chimeras successfully operated on both native att-sites and hybrid att-sites.
• CT chimeras were active only on hybrid att-sites.
• A BC hybrid (BC{-1}) exhibited activity in human HeLa cells.

Conclusions:

• This study presents the first demonstration of chimeric serine integrase activity.
• The findings provide insights into serine integrase structure-function relationships.
• The developed chimeric integrases offer a potentially tractable method for probing the specificity of numerous putative large serine recombinases identified through bioinformatics.