Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display.

Abstract:

: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.

journal_name

FEMS Microbiol Lett

authors

Kronqvist N,Löfblom J,Severa D,Ståhl S,Wernérus H

doi

10.1111/j.1574-6968.2007.00990.x

subject

Has Abstract

pub_date

2008-01-01 00:00:00

pages

128-36

issue

1

eissn

0378-1097

issn

1574-6968

pii

FML990

journal_volume

278

pub_type

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