Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS)

Abstract

Periplasmic expression with cytometric screening (PECS) is a powerful and rapid "display-less" technology for isolating ligand-binding proteins from diverse libraries. Escherichia coli expressing a library of proteins secreted into the periplasmic space are incubated with a fluorescent conjugate of the target ligand. Under the proper conditions, ligands as large as about 10 kDa can equilibrate within the periplasmic space without compromising the cell's integrity or viability. The bacterial cell envelope effectively serves as a dialysis bag to selectively retain receptor-fluorescent probe complexes but not free ligand. Cells displaying increased fluorescence are then isolated by flow cytometry. We demonstrate that scFv antibodies with both very high and low affinity to digoxigenin can be isolated from libraries screened by PECS using a benchtop flow cytometer. We also show that preexisting libraries constructed for display on filamentous bacteriophage can be screened by PECS without the need for subcloning. In fact, PECS was found to select for proteins that could be missed by conventional phage panning and screening methods.

Tailoring in Vitro Selection for a Picomolar Affinity Human Antibody Directed against Vascular Endothelial Growth Factor Receptor 2 for Enhanced Neutralizing Activity

“…A variety of methods, including randomized mutations (33)(34)(35), designed mutations such as "hot spot" and "codon-based" mutations (guided by structural knowledge and/or computer modeling) (36 -38) and "CDR walking" (39) have been used to construct the mutagenized libraries. These libraries were then displayed on the surface of filamentous phage (40,41), bacterial (42)(43)(44), yeast (45,46), or ribosome (47-49) and selected on relevant targets under conditions specifically designed for the enrichment of variants with desired properties (i.e. high affinity) (for review, see Ref.…”

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

“…These include, for instance, phage display, retroviral display, bacterial display, yeast display and various mammalian cell display technologies, in combination with solid surface binding (panning) or other enrichment techniques. 2,3 While phage, 4-6 prokaryotic 7,8 and ribosome/mRNA display 9,10 systems have been established and are widely adopted in the biotechnology industry and in academia for the identification of antibody fragments, they suffer from a variety of limitations, including the inability to express full-length antibodies, the absence of natural post-translational modifications, the lack of proper folding by vertebrate chaperones, and an artificially enforced heavy and light chain combination. Therefore, the "reformatting" of antibody fragments into full-length antibodies followed by manufacturing in mammalian cells frequently results in molecules with unfavorable biophysical properties (e.g., low stability, tendency to aggregate, diminished affinity).…”

In Vivo Imaging of a Bacterial Cell Division Protein Using a Protease‐Assisted Small‐Molecule Labeling Approach

“…[1] Since the outer membrane of Gram negative bacteria restricts the diffusion of molecules larger than ~ 650 Da, we kept the size of our probes within this molecular weight limit. [2] 3. In vitro labeling of benzyl-thioester probe with cysteine proteases…”