Abstract
The first demonstration of wheat germ extract (WGE)-based in vitro translation synthesising a protein from exogenously introduced messenger ribonucleic acid (mRNA) was published approximately fifty years ago. Since then, there have been numerous crucial improvements to the WGE-based in vitro translation, resulting in a significant increase in yield and the development of high-throughput protein-producing platforms. These developments have transformed the original setup into a versatile eukaryotic protein production method with broad applications. The present review explores the theoretical background of the implemented modifications and brings a panel of examples for WGE applications in high-throughput protein studies and synthesis of challenging-to-produce proteins such as protein complexes, extracellular proteins, and membrane proteins. It also highlights the unique advantages of in vitro translation as an open system for synthesising radioactively labelled proteins, as illustrated by numerous publications using WGE to meet the protein demands of these studies. This review aims to orientate readers in finding the most appropriate WGE arrangement for their specific needs and demonstrate that a deeper understanding of the system modifications will help them make further adjustments to the reaction conditions for synthesising difficult-to-express proteins.