Selecting high-affinity binding proteins by monovalent phage display.
Variants of human growth hormone (hGH) with increased affinity and specificity for the hGH receptor were isolated using an improved phage display system. Nearly one million random mutants of hGH were generated at 12 sites previously shown to modulate binding to the hGH receptor or human prolactin (hPRL) receptor. The mutant hormones were displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. After three to six cycles of enrichment for hGH-phage particles that bound to hGH receptor beads, we isolated hGH mutants that exhibited consensus binding sequences for the hGH receptor. Residues previously identified as important for hGH receptor binding by alanine-scanning mutagenesis were more highly conserved by this selection method. However, other residues nearby were not optimal, and by mutating them, hormone variants having greater affinity and selectivity for the hGH receptor were isolated. This approach should be useful for those who wish to modify and understand the energetics of protein-ligand interfaces.
Lowman
H B
HB
Department of Protein Engineering, Genetech, Inc., South San Francisco, California 94080.
Bass
S H
SH
Simpson
N
N
Wells
J A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism; D003062:Codon / Q000235:genetics; D003090:Coliphages / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D013006:Growth Hormone / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011487:Protein Conformation; D011986:Receptors, Somatotropin / Q000378:metabolism*; D015183:Restriction Mapping
D002352:Carrier Proteins; D003062:Codon; D009838:Oligodeoxyribonucleotides; D011986:Receptors, Somatotropin; D013006:Growth Hormone