Open AccessSimplified characterization through site‐specific protease‐mediated release of affinity proteins selected by staphylococcal display
Author(s) -
Kronqvist Nina,
Löfblom John,
Severa Denise,
Ståhl Stefan,
Wernérus Henrik
Publication year - 2008
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2007.00990.x
Subject(s) - protease , affinity chromatography , cleavage (geology) , fusion protein , biochemistry , tandem affinity purification , protein purification , chemistry , biology , computational biology , enzyme , recombinant dna , gene , paleontology , fracture (geology)
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.