Advances and Challenges in Cell-Free Incorporation of Unnatural Amino Acids Into Proteins.

Wei Gao,Yingying Liu,Yuan Lu,Eunhee Cho

doi:10.3389/fphar.2019.00611

Abstract

Incorporation of unnatural amino acids (UNAAs) into proteins currently is an active biological research area for various fundamental and applied science. In this context, cell-free synthetic biology (CFSB) has been developed and recognized as a robust testing and biomanufacturing platform for highly efficient UNAA incorporation. It enables the orchestration of unnatural biological machinery toward an exclusive user-defined objective of unnatural protein synthesis. This review aims to overview the principles of cell-free unnatural protein synthesis (CFUPS) systems, their advantages, different UNAA incorporation approaches, and recent achievements. These have catalyzed cutting-edge research and diverse emerging applications. Especially, present challenges and future trends are focused and discussed. With the development of CFSB and the fusion with other advanced next-generation technologies, CFUPS systems would explicitly deliver their values for biopharmaceutical applications.

Highlights

The incorporation of unnatural amino acids (UNAAs) in protein engineering has emerged as a key discipline in synthetic biology
Besides direct chemical reaction with the side chains of standard amino acids (SAAs) and the biological posttranslational modifications (PTMs) (Zhang et al, 2018), there has been an extensive research in making unnatural amino acids (UNAAs) with novel characteristics and incorporating them into proteins during translation (Liu and Schultz, 2010; Chin, 2017)
Conventional cell-based in vivo systems are facing many engineering challenges, including the UNAA transportation cross the cell membrane, the cytotoxicity of unnatural biological components, and low incorporation efficiency

Summary

INTRODUCTION

The incorporation of unnatural amino acids (UNAAs) in protein engineering has emerged as a key discipline in synthetic biology. Conventional cell-based in vivo systems are facing many engineering challenges, including the UNAA transportation cross the cell membrane, the cytotoxicity of unnatural biological components, and low incorporation efficiency. In this context, the emerging cell-free unnatural protein synthesis (CFUPS) systems have been adopted to break through those restrictions. CFUPS systems as good high-throughput screening platforms can speed up the design–build–test cycle for unnatural protein engineering (Mankowska et al, 2016) All these advantages have transformed the CFUPS system into a robust UNAA incorporation platform for the synthesis of unnatural proteins with great simplicity and flexibility. The crude extract-based CFUPS system is preferred for applied research

UNNATURAL AMINO ACID INCORPORATION APPROACHES

EMERGING APPLICATIONS

CHALLENGES AND FUTURE PROSPECTS

Findings

Orthogonal aaRS Orthogonal tRNA Orthogonal translation system