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

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.

Biomolecular solid-state NMR spectroscopy at 1200 MHz: the gain in resolution

“…First, perdeuteration followed by back-exchange of the amide protons (and exchangeable side-chain protons) is often used to improve resolution, so that solely H N are present in the sample. Selective labeling allows to introduce protons at selected side-chain positions (Kainosho et al 2006 ; Asami and Reif 2012 ; Lacabanne et al 2017 ; Movellan et al 2019 ; Fogeron et al 2021 ), but can make protein expression more complex and expensive. Detection of H and side-chain protons is of course possible in fully protonated samples, but with an increased line width, resulting in poorer sensitivity and resolution.…”

Lactococcus lactis, an Attractive Cell Factory for the Expression of Functional Membrane Proteins

“…Therefore, the low concentration of MPs in cells highlights the need for heterologous expression systems. There are different types of expression systems such as cell-free systems [ 6 ], prokaryotic systems ( E. coli and L. lactis ), and eukaryotic expression systems (yeasts, plants, mammalian, or insect cells). All of them have advantages and drawbacks [ 3 , 4 , 7 ].…”

Cell-free Protein Crystallization for Nanocrystal Structure Determination

“…At 15 °C and 25 °C, cubic crystals were observed, but many round crystals were observed at 4 °C and 10 °C. Although temperature is expected to affect the expression yield during translation reaction, the yields of PhM after 24 h did not differ significantly in the temperature range as confirmed by SDS-PAGE (Supplementary Figure 3b) 19 . Therefore, the crystal size and morphology are expected to be affected by the crystallization rate at various temperatures.…”

“…Cell-free protein synthesis (CFPS), a protein preparation technique used in synthetic biology, is very effective for rapid screening of protein synthesis 18 . However, it has been considered unsuitable for structural biology efforts that require large amounts of protein, such as crystallization [19][20][21] . Here, we report the application of CFPS to ICPC (Figure 1).…”