Abstract
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.
Highlights
Protein engineering is a technology that tailors a protein to function in a desired way
We introduce a new liposome-based In vitro compartmentalization (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
The protein variants are synthesized from the mutated genes using living hosts or an in vitro transcription-translation system (IVTT), and they are screened for the desired function
Summary
Protein engineering is a technology that tailors a protein to function in a desired way. 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. Directed evolution of proteins is based on Darwinian selection and 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]. The methods used for directed evolution can be categorized as “in vivo” and “in vitro” approaches
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
