Title:
Approaches to improve expression and specificity of an antibody probe against fibronectin

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Moretti, Leandro
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Barker, Thomas H.
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Abstract
Phage display is a convenient method to select proteins of interest based on binding affinity. The Barker lab recently used this technology to discover an antibody fragment (scFv), termed H5, capable of selectively binding to the integrin binding domain of fibronectin (Fn) under a strained configuration, typically due to forces exerted on the extracellular matrix (ECM) by cells. Large scale production of H5 scFv is hindered by non-optimized codons and suspected protein toxicity, since the E. coli strain producing the protein grew poorly at normal temperatures. Moreover, the transfected vector containing H5, pIT2, appeared to be degraded in consecutively selected bacterial cultures, as suggested by the isolation of significantly shorter than expect plasmids. After significant effort, reconstruction of the pIT2 vector sequence was accomplished from multiple colonies and cross referencing with the provided anti-ubiquitin scFv antibody fragment, and the complete DNA sequence of H5 was recovered. The sequence was codon optimized for expression in an E. coli strain engineered for high levels of scFv production. Furthermore, the H5 DNA was recombined with error prone PCR, to generate a library of random mutants, which was transformed into a stable, E. coli strain. This library will be panned on the targets again through phage display in order to find an improved version of H5, defined by more selective binding to the extended conformation of the integrin binding domain of Fn.
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2016-08-30
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