Protein and Antibody Engineering by Phage Display.

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process.

Toward beta-peptide tertiary structure: self-association of an amphiphilic 14-helix in aqueous solution.

A major frontier in foldamer research is creation of unnatural oligomers that adopt discrete tertiary structures; at present, only biopolymers are known to fold into such compact conformations. We report an initial step toward helix-bundle tertiary structure in the beta-peptide realm by showing that a 10-residue beta-peptide designed to adopt an amphiphilic helical conformation forms small soluble aggregates in water. Sedimentation equilibrium data indicate that the aggregated state falls in the tetramer-hexamer size range. [structure: see text]

Enhancement of tyrosyl phosphorylation and protein expression of eps8 by v-Src.

Two eps8 isoforms, p97eps8 and p68eps8, were previously identified as substrates for receptor tyrosine kinases. Analysis of eps8 phosphotyrosine content in v-Src transformed cells (IV5) revealed that both isoforms were highly tyrosyl phosphorylated and their readiness to be phosphorylated by Src in vitro further indicated that they were putative Src substrates as well. Indeed, the enhancement of tyrosyl phosphorylation of p97eps8 detected in cells coexpressing both p97eps8 and active Src relative to that in cells expressing p97eps8 alone supported our hypothesis. The existence of common phosphotryptic peptides between in vitro 32P-labeled p97eps8 and p68eps8 indicated that these two proteins shared the same Src-mediated sites. Further in vitro binding assays demonstrated that p68eps8 was the major eps8 isoforms that could be precipitated by bacterial fusion protein containing Src SH3. Interestingly, both p68eps8 and p97eps8 were preferentially expressed in v-Src transformed cells and the presence of p68eps8 appeared to depend on Src. Since p97eps8 has been implicated in mitogenesis and tumorigenesis, its readiness to be phosphorylated and induced by v-Src might attribute to v-Src-mediated transformation.