The Application of Cell-Free Protein Synthesis in Genetic Code Expansion for Post-translational Modifications.

Sumana Venkat,Chenguang Fan,Hao Chen,Qinglei Gan

doi:10.3389/fphar.2019.00248

Sumana Venkat, Chenguang Fan + Show 2 more

Open Access

https://doi.org/10.3389/fphar.2019.00248

Copy DOI

Abstract

The translation system is a sophisticated machinery that synthesizes proteins from 20 canonical amino acids. Recently, the repertoire of such composition has been expanded by the introduction of non-canonical amino acids (ncAAs) with the genetic code expansion strategy, which provides proteins with designed properties and structures for protein studies and engineering. Although the genetic code expansion strategy has been mostly implemented by using living cells as the host, a number of limits such as poor cellular uptake or solubility of specific ncAA substrates and the toxicity of target proteins have hindered the production of certain ncAA-modified proteins. To overcome those challenges, cell-free protein synthesis (CFPS) has been applied as it allows the precise control of reaction components. Several approaches have been recently developed to increase the purity and efficiency of ncAA incorporation in CFPS. Here, we summarized recent development of CFPS with an emphasis on its applications in generating site-specific protein post-translational modifications by the genetic code expansion strategy.

Highlights

The translational machinery in nature provides a new avenue of chemistry in which synthetic biologists could prospect beyond the repertoire of 20 canonical amino acids (Liu and Schultz, 2010)
We summarized the applications of cell-free protein synthesis (CFPS) in generating proteins with post-translational modifications (PTMs) through the genetic code expansion strategy
By using vasodilator-stimulated phosphoprotein (VASP) that is involved in cell migration processes as a target protein, the phosphoprotein generated by CFPS was shown to be active as expected

Summary

INTRODUCTION

The translational machinery in nature provides a new avenue of chemistry in which synthetic biologists could prospect beyond the repertoire of 20 canonical amino acids (Liu and Schultz, 2010). Because of diversified functional groups, ncAAs endow proteins with modified structures, functions, and interactions (O’Donoghue et al, 2013) This approach, named genetic code expansion, has paved the way for various applications in biological studies (Chin, 2014; Li and Liu, 2014; Neumann-Staubitz and Neumann, 2016). It permits the synthesis of proteins toxic to cells (Worst et al, 2015) It has no limit for cellular uptake and solubility of target ncAAs (Bundy and Swartz, 2010). Competitions among different PTMs for the same residues and limited knowledge of modifying enzymes make it difficult to synthesize homogeneously modified proteins for studying To overcome these challenges, the genetic code expansion strategy has been applied in PTM studies (Liu et al, 2011; Chen et al, 2018). We summarized the applications of CFPS in generating proteins with PTMs through the genetic code expansion strategy

Tyrosine Phosphorylation

Serine Phosphorylation

Lysine Acetylation

Lysine Methylation

CONCLUSIONS