2016
DOI: 10.3791/54583
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Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Abstract: Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to s… Show more

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Cited by 6 publications
(4 citation statements)
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“…The binding partner of an antibody is usually really large and, thus, can not permeate the outer cell membrane of the E. coli like in our case where we deal with small molecules (asparagine and aspartate). Hence, for antibody engineering the display approach would be really ideal in order to expose the anchored antibody fragment upon outer membrane permeabilization for binding studies. Aiming at the improvement of the kinetics and, in turn, of the signal-to-noise ratio of the display approach, in a set of experiments we measure the reaction rate of our assay in droplets of volumes ranging from 800 pL down to 50 pL. By decreasing approximately 16-fold the volume of the droplets (from 800 pL to 50 pL), we achieve a considerable improvement of the kinetics of ∼3-fold (Figure A).…”
Section: Resultsmentioning
confidence: 99%
“…The binding partner of an antibody is usually really large and, thus, can not permeate the outer cell membrane of the E. coli like in our case where we deal with small molecules (asparagine and aspartate). Hence, for antibody engineering the display approach would be really ideal in order to expose the anchored antibody fragment upon outer membrane permeabilization for binding studies. Aiming at the improvement of the kinetics and, in turn, of the signal-to-noise ratio of the display approach, in a set of experiments we measure the reaction rate of our assay in droplets of volumes ranging from 800 pL down to 50 pL. By decreasing approximately 16-fold the volume of the droplets (from 800 pL to 50 pL), we achieve a considerable improvement of the kinetics of ∼3-fold (Figure A).…”
Section: Resultsmentioning
confidence: 99%
“…These include yeast display, whereby single-chain variable fragments (scFvs) are displayed on the surface of yeast cells [8]; mRNA display [9]; ribosome display, which integrates a ribosome, mRNA and an scFv [10]; bacterial inner-membrane display [11]; and mammalian cell surface display [12] for human antibody discovery. In phage display, antibody fragments are joined to the filamentous phage coat protein g3p.…”
mentioning
confidence: 99%
“…However, this export is reliant on the recombinant protein refolding to a native, biologically active conformation in the periplasm. Such an approach has previously been used when displaying scFVs via Tat machinery (Karlsson et al, 2012;Moghaddam-Taaheri, Ikonomova, Gong, Wisniewski, & Karlsson, 2016). Whereas inner membrane display does require outer membrane removal to gain access to the recombinant protein, in instances where cell surface display on the outer membrane of bacteria is impossible, or hugely inefficient, this "extra-step" of membrane removal far outweighs the difficulty of displaying protein at the outer membrane.…”
mentioning
confidence: 99%
“…Whereas inner membrane display does require outer membrane removal to gain access to the recombinant protein, in instances where cell surface display on the outer membrane of bacteria is impossible, or hugely inefficient, this "extra-step" of membrane removal far outweighs the difficulty of displaying protein at the outer membrane. Such an approach has previously been used when displaying scFVs via Tat machinery (Karlsson et al, 2012;Moghaddam-Taaheri, Ikonomova, Gong, Wisniewski, & Karlsson, 2016). This inherent quality control mechanism of the Tat machinery, in combination with data showing that a signal peptide mutation in a Tat substrate is able to stall a Tat precursor at the inner membrane (Ren et al, 2013), highlights the potential for Tat machinery in cell surface display technology.…”
mentioning
confidence: 99%