Hydrophobic supplements in cell‐free systems: Designing artificial environments for membrane proteins

Abstract

Membrane proteins (MPs) are of central interest for the pharmaceutical industry but their production is usually a challenging task. The complex folding mechanisms of newly synthesized MPs often require interactions with specific compounds for improved stability. Conditions for the production of high-quality samples are therefore difficult to predict and frequently cannot be provided in conventional protein expression platforms. Cell-free (CF) biosynthetic systems allow, in contrast to living cells, nonrestricted access to the protein production machinery. Reaction conditions can be adjusted according to particular requirements and modified by supplementing single additives or even cocktails of compounds. These options have initiated completely new research fields for the cotranslational stabilization and folding of MPs in artificial environments. Based on established and efficient CF production protocols, a recent focus was to explore and define suitable supplements for CF expression reactions that are useful for the generation of high-quality MP samples. Besides classical detergents and lipids, a variety of new compounds with interesting properties have been discovered and synthesized. We compile the currently available toolbox for MP solubilization in CF systems and summarize new developments and perspectives for the directed modulation of CF biosynthetic environments.

Nanodiscs in Membrane Biochemistry and Biophysics

“…Self-assembly into Nanodiscs restores the native state. 272,273 There are a plethora of spectroscopic investigations that have successfully used Nanodiscs. Nanodiscs provided especially significant advantages for spectroscopic investigations due to their lack of turbidity and low viscosity.…”

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

“…MurA, -B, -D, -E, and -F were expressed with a C-terminal poly(His) 6 tag for purification and detection. The MurC and MurG proteins were produced with a N-terminal poly(His) 6 tag and a C-terminal StrepII tag or a C-terminal poly(His) 10 tag, respectively (Table 1). All enzymes were synthesized in standard CECF reactions without additives.…”

“…Generally, membrane proteins could either be solubilized in the presence of micelles formed by amphiphilic compounds (D-CF, detergent-based cell-free expression) or in the presence of membranes of defined or complex lipid compositions (L-CF, lipidbased cell-free expression). In particular, the combination of the nanodisc technology with cell-free expression opens promising perspectives for the systematic screening of lipid effects on the functional folding of membrane proteins (10,11). If synthesized without any supplied hydrophobic environment, membrane proteins will precipitate (P-CF, precipitate forming cellfree expression) and often form type I aggregates that may become post-translationally solubilized into functional proteins (12)(13)(14).…”

Screening for Lipid Requirements of Membrane Proteins by Combining Cell-Free Expression with Nanodiscs

“…Even complex eukaryotic membrane proteins or multi protein complexes could be synthesized in functionally folded conditions by this procedure Keller et al, 2011;Matthies et al, 2011). Alternatively, hydrophobic agents can be supplied directly into the reactions for the cotranslational solubilization of the synthesized membrane proteins in the detergent-based D-CF expression mode (Hein et al, 2014). The list of D-CF suitable hydrophobic additives is continuously growing, and in particular, compound cocktails may show synergies in membrane protein solubilization, stabilization, and folding (Nozawa et al, 2007).…”

“…Stabilizing agents such as chemical chaperones or amphiphilic compounds could therefore be provided in almost any combination (Hein, Henrich, Orbán, D€ otsch, & Bernhard, 2014). In particular, the role of lipids as folding chaperones for membrane proteins becomes increasingly recognized (Hunte & Richers, 2008).…”