Hindawi Publishing Corporation Journal of Nucleic Acids Volume 2012, Article ID 923214, 11 pages doi:10.1155/2012/923214 Review Article Directed Evolution of Proteins through In Vitro Protein Synthesis in Liposomes Takehiro Nishikawa,1 Takeshi Sunami,1, 2 Tomoaki Matsuura,1, 3 and Tetsuya Yomo1, 2, 4 1 ERATO Japan Science and Technology (JST) and Yomo Dynamical Micro-Scale Reaction Environment Project, Graduate School of Information Sciences and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan 2 Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan 3 Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan 4 Graduate School of Frontier Biosciences, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan Correspondence should be addressed to Tetsuya Yomo, yomo@ist.osaka-u.ac.jp Received 8 June 2012; Accepted 10 July 2012 Academic Editor: Hiroshi Murakami Copyright © 2012 Takehiro Nishikawa et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Directed evolution of proteins is a technique used to modify protein functions through “Darwinian selection.” In vitro compartmentalization (IVC) is an in vitro gene screening system for directed evolution of proteins. IVC establishes the link between genetic information (genotype) and the protein translated from the information (phenotype), which is essential for all directed evolution methods, by encapsulating both in a nonliving microcompartment. Herein, we introduce a new liposome-based IVC system consisting of a liposome, the protein synthesis using recombinant elements (PURE) system and a ﬂuorescence-activated cell sorter (FACS) used as a microcompartment, in vitro protein synthesis system, and high-throughput screen, respectively. Liposome- based IVC is characterized by in vitro protein synthesis from a single copy of a gene in a cell-sized unilamellar liposome and quantitative functional evaluation of the synthesized proteins. Examples of liposome-based IVC for screening proteins such as GFP and β-glucuronidase are described. We discuss the future directions for this method and its applications. 1. Introduction Protein engineering is a technology that tailors a protein to function in a desired way. Rational design and directed evolution are two major approaches for introducing a change into the amino acid sequence of proteins. As a small change in the protein sequence can induce critical functional changes in proteins, altering the amino acid sequence is a crucial step in these approaches; the amino acid sequences are primarily altered by introducing mutations in the gene that encodes the protein of interest. In site-directed mutagen- esis, speciﬁc mutations to the DNA sequence are introduced, which yields a desired function if the relationship between protein structure and function is clearly understood. How- ever, directed evolution of proteins is based on Darwinian selection and thus does not necessarily require knowledge of the relationship between protein sequence and function [1, 2]. Using this method, mutations are generated through techniques such as random mutagenesis, recombination, or site-directed diversiﬁcation [3]. Subsequently, the protein variants are synthesized from the mutated genes using living hosts (cells) or an in vitro transcription-translation system (IVTT), and they are screened for the desired function. Therefore, the methods used for directed evolution can be categorized as “in vivo” and “in vitro” approaches. The diﬀerence between these two approaches (in vivo and in vitro approach) is the way that the genotype (genetic information encoding a protein) and a phenotype (the protein synthesized from the gene and its function) are linked for the genes of interest (Figure 1). Through an in vivo approach, the genotype-phenotype link is produced by using a living cell. For example, cell-surface display is an in vivo screening technique that uses a fusion protein to localize protein molecules to a cell membrane surface. Target proteins fused with a membrane protein are displayed on the cell membrane surface, screened for the desired function by