Editor: Jan-Ingmar Flock
Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display
Article first published online: 22 NOV 2007
FEMS Microbiology Letters
Volume 278, Issue 1, pages 128–136, January 2008
How to Cite
Kronqvist, N., Löfblom, J., Severa, D., Ståhl, S. and Wernérus, H. (2008), Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display. FEMS Microbiology Letters, 278: 128–136. doi: 10.1111/j.1574-6968.2007.00990.x
- Issue published online: 22 NOV 2007
- Article first published online: 22 NOV 2007
- Received 29 August 2007; accepted 9 October 2007.First published online December 2007.
- combinatorial protein engineering;
- Gram-positive bacterial display;
- protein production;
- Staphylococcus carnosus
The production of candidate affinity proteins in a soluble form, for downstream characterization, is often a time-consuming step in combinatorial protein engineering methods. Here, a novel approach for efficient production of candidate clones is described based on direct cleavage of the affinity protein from the surface of Staphylococcus carnosus, followed by affinity purification. To find a suitable strategy, three new fusion protein constructs were created, introducing a protease site for specific cleavage and purification tags for affinity chromatography purifications into the staphylococcal display vector. The three modified strains were evaluated in terms of transformation frequency, surface expression level and protease cleavage efficiency. A protocol for efficient affinity purification of protease-released affinity proteins using the introduced fusion-tags was successfully used, and the functionality of protease-treated and purified proteins was verified in a biosensor assay. To evaluate the devised method, a previously selected HER2-specific affibody was produced applying the new principle and was used to analyze HER2 expression on human breast cancer cells.