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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Published on: March 16, 2011

Directed evolution of sortase A mutants with altered substrate selectivity profiles.

Kirill Piotukh¹, Bernhard Geltinger, Nadja Heinrich

¹Department of Protein Engineering, Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.

Journal of the American Chemical Society

|October 8, 2011

Summary

Researchers engineered a mutant sortase A enzyme with broad substrate selectivity using directed evolution. This breakthrough enables traceless protein semisynthesis, advancing protein engineering and biotechnological applications.

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Area of Science:

Biochemistry
Protein Engineering
Molecular Biology

Background:

Sortase A (SrtA) is a bacterial transpeptidase crucial for protein ligation in protein engineering.
Current SrtA methods necessitate the LPxTG motif, often introducing unwanted mutations during protein semisynthesis.
There is a need for sortase variants with altered substrate specificity for improved protein assembly.

Purpose of the Study:

To develop a mutant sortase A enzyme with broad substrate selectivity.
To enable traceless protein semisynthesis by overcoming the limitations of the LPxTG motif.
To establish a platform for engineered sortases with diverse ligation capabilities.

Main Methods:

Directed evolution was employed to create a mutant sortase A library.
Phage display screening was utilized to isolate sortase variants with altered substrate recognition loops.
The engineered sortase was applied for the traceless semisynthesis of histone H3.

Main Results:

A mutant sortase A with significantly broadened substrate selectivity was successfully isolated.
The engineered sortase facilitated the traceless semisynthesis of histone H3, eliminating the need for the LPxTG motif.
This represents a gain-of-function mutation enhancing sortase activity and versatility.

Conclusions:

Directed evolution of sortase A yields enzymes with tailored substrate specificity.
Traceless protein semisynthesis is achievable with engineered sortases, improving protein engineering efficiency.
This work paves the way for versatile protein assembly platforms using engineered sortases in biotechnology.