Hormone phage: an enrichment method for variant proteins with altered binding properties.
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.
Bass
S
S
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080.
Greene
R
R
Wells
J A
JA
D001483:Base Sequence; D015153:Blotting, Western; D002213:Capsid / Q000235:genetics*; D002846:Chromatography, Affinity; D003090:Coliphages / Q000235:genetics; D004252:DNA Mutational Analysis; D000939:Epitopes; D005822:Genetic Vectors; D013006:Growth Hormone / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000939:Epitopes; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone