Abstract
The selective immobilization of proteins represents an essential step in the selection of binding proteins such as antibodies. The immobilization strategy determines how the target protein is presented to the binders and thereby directly affects the experimental outcome. This poses specific challenges for membrane proteins due to their inherent lack of stability and limited exposed hydrophilic surfaces. Here we detail methodologies for the selective immobilization of membrane proteins based on the strong biotin-avidin interaction and with a specific focus on its application for the selection of nanobodies and sybodies. We discuss the challenges in generating and benefits of obtaining an equimolar biotin to target-protein ratio.
Keywords:
Avi-tag; Biotin; FX cloning; Nanobodies; Neutravidin; Panning; Phage display; Ribosome display; Streptavidin; Sybodies.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Avidin / chemistry
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Avidin / metabolism*
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Biotin / chemistry
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Biotin / metabolism*
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Biotinylation / methods*
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Carbon-Nitrogen Ligases / chemistry
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Carbon-Nitrogen Ligases / metabolism
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Cell Surface Display Techniques / methods
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Cloning, Molecular / methods
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Escherichia coli
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Escherichia coli Proteins / chemistry
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Escherichia coli Proteins / metabolism
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Klebsiella pneumoniae
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Membrane Proteins / chemistry
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Membrane Proteins / genetics
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Membrane Proteins / isolation & purification
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Membrane Proteins / metabolism*
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Protein Binding
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Repressor Proteins / chemistry
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Repressor Proteins / metabolism
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Ribosomes / chemistry
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Ribosomes / metabolism
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Single-Domain Antibodies / chemistry
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Single-Domain Antibodies / isolation & purification*
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Single-Domain Antibodies / metabolism
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Streptavidin / chemistry
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Streptavidin / metabolism
Substances
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Escherichia coli Proteins
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Membrane Proteins
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Repressor Proteins
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Single-Domain Antibodies
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Avidin
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Biotin
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Streptavidin
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Carbon-Nitrogen Ligases
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birA protein, E coli