T

Display sequence

Transcription and translation

Coat protein

Insert into phage coat

Coat protein III i

Coat protein III i

Phage particle

Displayed peptide

Phage particle

Displayed peptide

A mixture of viruses with different proteins displayed is screened for binding to an antibody or other specific binding protein.

produced are slightly longer than the original wild type M13; nonetheless, they are packaged by extending the filamentous protein coat. The fusion proteins are expressed and the intruding peptides displayed on the surface of the M13 particles.

Many proteins have regions that bind to other proteins. These regions can be large or small, but usually a few amino acids are critical for the two proteins to interact properly. In order to identify the peptide sequence these binding sites recognize, phage display libraries are constructed. These consist of a large number of modified phages displaying a library of different peptide sequences. The first such libraries consisted of large collections of short random peptides. They are screened to find peptides that bind to specific molecules (the "target"), such as a particular antibody, enzyme or cell-surface receptor. The peptide of interest is found by a selection procedure referred to as biopanning. The phage display library is incubated with target molecules that are attached to a solid support (beads or membranes, etc.). Unbound phage is washed away. Bound phage is eluted and amplified by re-infecting cells of E. coli. Several cycles of binding and amplification will enrich for the phage that carries the peptide that binds most tightly to the target. Finally, individual clones are characterized by DNA sequencing (Fig. 26.14).

Several commercially available peptide libraries are marketed by New England Biolabs under the trade name Ph.D. (for Phage .Display!).The Ph.D.-7 library consists of 2.0 x 109 random heptapeptide clones. It probably contains most of the theoretically possible amino acid heptamers (of which there are 207 = approximately 1.3 x 109). In contrast, the Ph.D.-12 library, also with 2.0 x 109 independent clones, only represents a small fraction of the 2012 = 4.1 x 101512-mers.

Full-length proteins can also be fused to phage coat proteins to produce a full-length phage display library. In principle, a gene library from any organism could be converted into a phage display library by insertion into a suitable phage coat protein gene. M13 is not practical for this type of library since the insert must be positioned between the signal sequence, which is needed for secretion and phage assembly, and biopanning Method of screening a phage display library for a desired displayed protein by binding to a bait molecule attached to a solid support phage display library Collection of a large number of modified phages displaying different peptide or protein sequences

A. Library of

B. Bind phage

C. Wash away

D. Release

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