%0 Journal Article
%T Practical Tips for Construction of Custom Peptide Libraries and Affinity Selection by Using Commercially Available Phage Display Cloning Systems
%A Keisuke Fukunaga
%A Masumi Taki
%J Journal of Nucleic Acids
%D 2012
%I Hindawi Publishing Corporation
%R 10.1155/2012/295719
%X Phage display technology is undoubtedly a powerful tool for affinity selection of target-specific peptide. Commercially available premade phage libraries allow us to take screening in the easiest way. On the other hand, construction of a custom phage library seems to be inaccessible, because several practical tips are absent in instructions. This paper focuses on what should be born in mind for beginners using commercially available cloning kits (Ph.D. with type 3 vector and T7Select systems for M13 and T7 phage, respectively). In the M13 system, Pro or a basic amino acid (especially, Arg) should be avoided at the N-terminus of peptide fused to gp3. In both systems, peptides containing odd number(s) of Cys should be designed with caution. Also, DNA sequencing of a constructed library before biopanning is highly recommended for finding unexpected bias. 1. Introduction Phage display technology was born in 1985 when George Smith reported that foreign peptide could be displayed on the surface of filamentous bacteriophage [3]. Today, the phage display is a versatile tool for finding specific interactions between randomized library peptides/proteins on phage and target proteins, peptides, or other molecules. For example, it is applicable for generation of therapeutic peptides against cancer [4], microbe [5], novel functional protein [6], or fully humanized monoclonal antibody [7]. The advantages of the phage display technology over other selection methods are as follows. (1) Cost of a routine is cheap. (2) Time required for selection/amplification is fast. (3) Extreme care for handling, such as RNA isolation/selection, is not necessary. The phage is a DNA-containing virus that infects bacteria and makes many copies of the library within a very short time [8]. A phage that specifically binds a target can be selected from mixtures of billions of phages, propagated by in vivo amplification, and then subjected to additional rounds of affinity selection (Figure 1). This whole process is so-called ˇ°biopanningˇ± [9]. After multiple rounds of the biopanning, enrichment of target-binding phage can be assessed by phage titering and enzyme-linked immunosorbent assay (ELISA). Finally, the peptide displayed on the phage can be analyzed by DNA sequencing. Figure 1: A typical procedure of the biopanning. (a) Incubation of phage library with an immobilized target. (b) Washing of unbound phage. (c) Elution of target-bound phage. (d) Amplification of the eluted phage for subsequent rounds of the biopanning. 1.1. Categorization of Phage Display Systems Based on vector systems,
%U http://www.hindawi.com/journals/jna/2012/295719/