Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology.

Marie-Laure Fogeron,Matthias Harbers,Laura Cole,Anja Böckmann,Lauriane Lecoq

doi:10.3389/fmolb.2021.639587

Abstract

Cell-free protein synthesis (CFPS) systems are gaining more importance as universal tools for basic research, applied sciences, and product development with new technologies emerging for their application. Huge progress was made in the field of synthetic biology using CFPS to develop new proteins for technical applications and therapy. Out of the available CFPS systems, wheat germ cell-free protein synthesis (WG-CFPS) merges the highest yields with the use of a eukaryotic ribosome, making it an excellent approach for the synthesis of complex eukaryotic proteins including, for example, protein complexes and membrane proteins. Separating the translation reaction from other cellular processes, CFPS offers a flexible means to adapt translation reactions to protein needs. There is a large demand for such potent, easy-to-use, rapid protein expression systems, which are optimally serving protein requirements to drive biochemical and structural biology research. We summarize here a general workflow for a wheat germ system providing examples from the literature, as well as applications used for our own studies in structural biology. With this review, we want to highlight the tremendous potential of the rapidly evolving and highly versatile CFPS systems, making them more widely used as common tools to recombinantly prepare particularly challenging recombinant eukaryotic proteins.

Highlights

Efficient, easy-to-use, and rapid protein expression methods for protein analysis are in great demand for structural determination, biochemical research, and applications in synthetic biology, such as the design of new biological circuits or the development of new proteins for technical applications and therapies (Gregorio et al, 2019; Silverman et al, 2020)
Cell-free protein synthesis (CFPS) was used to make versions of the SARS-CoV-2 N-protein using wheat germ cell-free protein synthesis (WG-CFPS) for use in serological testing (Matsuba et al, 2020; Yamaoka et al, 2020) and antibody development leading to tests for COVID-19 available on the market to serve patients
CFPS is getting more attention these days with the development of new methods that try to make the best use of the unique features of an in vitro method rather than relying on established systems depending on a host cell (Zemella et al, 2015) for synthesis of recombinant proteins

Summary

Introduction

Easy-to-use, and rapid protein expression methods for protein analysis are in great demand for structural determination, biochemical research, and applications in synthetic biology, such as the design of new biological circuits or the development of new proteins for technical applications and therapies (Gregorio et al, 2019; Silverman et al, 2020). The rapid response to the recent COVID-19 pandemic shows how the scientific community is applying the latest technologies to study viral proteins and to make them available for structural analysis (Zhu et al, 2020b) and drug testing (Dai et al, 2020; Jin et al, 2020), the development of antibodies, or creation of new serological tests to monitor infection rates. It is a promising approach to combine DNA detection with the expression of a marker protein, which will enable new concepts for biosensor developments (Duyen et al, 2016; Ogawa et al, 2016; Zhang et al, 2020) For such applications, the translation system could be miniaturized or used in a fluidic array device (Jackson et al, 2015) for automation and easy use

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