fulltext.study @t Gmail

Selection of TNF-α binding affibody molecules using a β-lactamase protein fragment complementation assay

Paper ID Volume ID Publish Year Pages File Format Full-Text
33706 44988 2009 9 PDF Available
Title
Selection of TNF-α binding affibody molecules using a β-lactamase protein fragment complementation assay
Abstract

Protein fragment complementation assays (PCAs) based on different reporter proteins have been described as powerful tools for monitoring dynamic protein–protein interactions in living cells. The present study describes the construction of a PCA system based on genetic splitting of TEM-1 β-lactamase for the selection of proteins specifically interacting in the periplasm of Escherichia coli bacterial cells, and its application for the selection of affibody molecules binding human tumour necrosis factor-α (TNF-α) from a combinatorial library. Vectors encoding individual members of a naïve 109 affibody protein library fused to a C-terminal fragment of the β-lactamase reporter were distributed via phage infection to a culture of cells harbouring a common construct encoding a fusion protein between a non-membrane anchored version of a human TNF-α target and the N-terminal segment of the reporter. An initial binding analysis of 29 library variants derived from surviving colonies using selection plates containing ampicillin and in some cases also the β-lactamase inhibitor tazobactam, indicated a stringent selection for target binding variants. Subsequent analyses showed that the binding affinities (KD) for three selected variants studied in more detail were in the range 14–27 nm. The selectivity in binding to TNF-α for these variants was further demonstrated in both a cross-target PCA-based challenge and the specific detection of a low nm concentration of TNF-α spiked into a complex cell lysate sample. Further, in a biosensor-based competition assay, the binding to TNF-α of three investigated affibody variants could be completely blocked by premixing the target with the therapeutic monoclonal antibody adalimumab (Humira®), indicating overlapping epitopes between the two classes of reagents. The data indicate that β-lactamase PCA is a promising methodology for stringent selection of binders from complex naïve libraries to yield high affinity reagents with selective target binding characteristics.

First Page Preview
Selection of TNF-α binding affibody molecules using a β-lactamase protein fragment complementation assay
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us
Publisher
Database: Elsevier - ScienceDirect
Journal: New Biotechnology - Volume 26, Issue 5, 30 November 2009, Pages 251–259
Authors
, , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us