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Updated: Jun 27, 2026
12:02
Molecular Evolution of the Tre Recombinase
Published on: May 29, 2008
Molecular evolution of the tre recombinase
1Max Plank Institute for Molecular Cell Biology and Genetics, Dresden. buchholz@mpi-cbg.de
Journal of Visualized Experiments : Jove
|December 11, 2008
Summary
Researchers engineered a novel enzyme, Tre recombinase, using directed molecular evolution to precisely excise HIV-1 provirus from infected human cells, offering a potential new tool for molecular medicine.
Area of Science:
- Molecular Biology
- Biotechnology
- Virology
Background:
- The human immunodeficiency virus type 1 (HIV-1) integrates into the host genome, establishing a persistent infection.
- Existing therapeutic strategies primarily focus on managing viral replication rather than eradicating the integrated provirus.
- The Cre recombinase system, while effective for genomic manipulation, does not naturally target HIV-1 sequences.
Purpose of the Study:
- To engineer a novel recombinase enzyme capable of recognizing and excising integrated HIV-1 proviral DNA.
- To adapt the Cre recombinase system for targeted removal of viral sequences from the human genome.
- To explore the potential of directed molecular evolution for creating custom enzymes for therapeutic applications.
Main Methods:
- Directed molecular evolution was employed to modify the Cre recombinase.
- Initial screening involved identifying and testing sequences within HIV-1 LTR sites similar to the Cre target sequence (loxP).
- Iterative cycles of enrichment, shuffling, and recombination of mutagenized recombinase libraries were performed to enhance target specificity and activity.
Main Results:
- Initial attempts with Cre and mutagenized libraries failed to recombine the target HIV-1 LTR sequences.
- Recombination activity was observed with intermediate sequence subsets, validating the evolutionary strategy.
- After extensive evolution (126 cycles), the Tre recombinase was generated, exhibiting significant activity.
- Tre recombinase, with 19 amino acid changes from Cre, successfully excised HIV-1 provirus from infected HeLa cells.
Conclusions:
- Directed molecular evolution is a viable strategy for creating custom enzymes with novel DNA recognition and recombination capabilities.
- Tre recombinase demonstrates the potential for targeted excision of integrated HIV-1 provirus, representing a significant advancement in potential HIV eradication strategies.
- Engineered recombinases hold promise as tools for 'molecular surgery' in the field of molecular medicine.
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