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 mutagenesis, specific mutations to the DNA sequence are introduced, which yields a desired function if the relationship between protein structure and function is clearly understood. However, 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
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