Phage

FIGURE 26.14 Biopanning to Screen a Phage Display Library

FIGURE 26.14 Biopanning to Screen a Phage Display Library

Biopanning is used to isolate peptides that bind to a specific target protein, which is usually attached to a solid support such as a membrane or column. The phage display library (A) is added to the binding protein (B). Those phage which display peptides that bind to the target protein will be retained (C) but the others are washed away. The phage that does recognize the binding protein can then be released, isolated and purified.

the N-terminus of the coat protein. Therefore, both ends of the insert must thus be in frame, and in addition, no stop codons must be present in the insert. In contrast, in T7 the C-terminal region of the coat protein is exposed on the outside. Using C-terminal coat protein fusions avoids the above problems and allows complete coding sequences to be inserted. Using the T7SelectTM system from Novagen, several cDNA libraries have now been biopanned to find proteins that bind to some chosen target molecule. For example, phage that display RNA-binding proteins have been isolated using RNA anchored to a solid support as bait (Fig. 26.15).

Other display systems use whole bacterial cells to carry the protein of interest. DNA sequences encoding polypeptides to be screened can be fused to the flagellin or pilin genes of E coli. The polypeptide library is then exposed on the cell surface attached to either the flagella or the pili. The phage display libraries are generally more convenient but one advantage of using bacteria is that the fluorescence-activated cell sorter (FACS) can sort the cells provided that the peptide target is labeled with a fluorescent dye.

Protein Interactions: The Yeast Two-Hybrid System

Many proteins recognize and bind to other proteins. The total of all protein-protein interactions is sometimes referred to as the protein interactome by those enthusiastic about "omics" terminology. Mass screening of such interactions has proven possible by means of the "two-hybrid" system. Interactome analysis is based on the idea of guilt protein interactome The total of all the protein-protein interactions in a particular cell or organism two-hybrid system Method of screening for protein-protein interactions that uses fusions of the proteins being investigated to the two separate domains of a transcriptional activator protein

DNA gene^OB RNA-BP gene

Packaging

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