Hormone phage: An enrichment method for variant proteins with altered binding properties

Abstract

Human growth hormone (hGH), a 191 residue protein containing two disulfide bonds, was fused to the carboxyl-terminal domain of the gene III protein, a minor coat protein exposed at one end of the filamentous phage M13. The gene fusion was cloned into a plasmid containing origins of replication for Escherichia coli and filamentous phage and was packaged into phagemid particles upon infection by an M13KO7 helper phage. Transcription of the hGH-gene III fusion was controlled so that usually no more than one copy of the fusion protein was displayed along with the four copies of the wild-type gene III protein. The hGH-gene III fusion protein was properly folded, as judged by reactivity with six hGH monoclonal antibodies whose epitopes are sensitive to the folded conformation of hGH. Moreover, the hGH-gene III phagemid particles were enriched over 5000-fold from non-hGH phage, and 8-fold from a mutant hGH phagemid following a single hGH-specific elution step from hGH receptor-coated beads. The hGH phagemid should be useful for isolating new receptor binding mutants of hGH. More generally, this expression system may allow other large proteins with discontinuous binding epitopes to be displayed, and binding selections applied to their mutated gene III fusions on filamentous phage.

Abrogation of Viral Interleukin-6 (vIL-6)-Induced Signaling by Intracellular Retention and Neutralization of vIL-6 with an Anti-vIL-6 Single-Chain Antibody Selected by Phage Display

“…Phage display technology provides a means by which high-affinity, fully human monoclonal antibodies can be rapidly isolated and also provides a starting point from which a selected antibody can, if necessary, be affinity maturated for improved neutralization potency or binding kinetics (6,41,73).…”

Phage display reveals 52 novel extracellular and transmembrane proteins from Lactobacillus reuteri DSM 20016T

“…Usually, the fusion protein is expressed with a phagemid vector, i.e. a plasmid containing a filamentous phage intergenic region, and a helper phage (Bass et al, 1990). The vector encodes the signal peptide required for direction of the fusion protein to the cell membrane, where phage assembly occurs.…”

Phage display mutagenesis of the chimeric dual cytokine receptor agonist myelopoietin

“…The mean activity of the selectants increased with the number of rounds of biopanning (data not shown), suggesting that receptor affinity/bioactivity maturation 11,12,16 resulted from the affinity selection. The biological activities of the variant myelopoietins were separated into three classes.…”

“…We used phage display mutagenesis as it allows survey of orders of magnitude more variants than conventional methods. [11][12][13] Several four-helical bundle proteins 2 have been presented on phage, [14][15][16][17][18][19] though chimeras containing multiple cytokines have not. Still, if both chimera domains are presented in functional conformations, display mutagenesis should be applicable to myelopoietin.…”