High copy display of large proteins on phage for functional selections.
We have isolated mutations in the major coat protein P8 of M13 phage that greatly increase the surface display of monomeric or oligomeric proteins. The monomeric protein, human growth hormone (hGH), was fused to the N terminus of P8; libraries of P8 variants were constructed and variants that increased hGH display were selected by binding to the extracellular domain of the hGH receptor. The hGH-P8 fusion protein was found to be extremely tolerant of mutations, and a number of P8 variants were found that increased display to levels that improved detection of the hGH-P8 fusion by almost 100-fold. The increased display likely results from better accommodation of the hGH-P8 fusion protein in the phage coat. Using this high copy display format, it was possible for the first time to detect variants of hGH with very weak affinities for the hGHbp (K(d)>1 microM). The display of a tetrameric protein, streptavidin (approximately 50 kDa), was also increased, suggesting the approach may be general to many proteins. The initial product of a natural or invented selection from a naive library is often a weakly functioning protein. These improvements in high copy display should facilitate the broader goal for selection of proteins with novel functions.
Sidhu
S S
SS
Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080, USA.
Weiss
G A
GA
Wells
J A
JA
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D001483:Base Sequence; D002213:Capsid / Q000235:genetics / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D014644:Genetic Variation / Q000235:genetics; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011986:Receptors, Somatotropin / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D013816:Thermodynamics
D007106:Immune Sera; D010446:Peptide Fragments; D019151:Peptide Library; D011506:Proteins; D011986:Receptors, Somatotropin; D011993:Recombinant Fusion Proteins; D019382:Human Growth Hormone; D019809:Streptavidin