Cell-Free Production of Integral Membrane Proteins on a Preparative Scale

Recent advances in the application of solution NMR spectroscopy to multi-span integral membrane proteins

“…Thus obtaining recombinant IMPs using exogenous expression is often a necessity in order to obtain adequate levels of protein production for structural studies. A number of reviews of IMP expression and sample preparation are available [22, 26, 28-45]. In the following section, several of the common expression systems for producing viable solution NMR samples will be explored, which are summarized in Table 1.…”

“…Recently, cell-free systems have emerged as a promising alternative for preparing large quantities of isotopically labeled membrane proteins [25, 26, 30, 33, 81, 82]. The application of a cell-free system offers some advantages over conventional expression of membrane proteins: (i) this approach is independent of cellular integrity, (ii) many different conditions can be tested to optimize expression levels in a short period of time, (iii) labeled proteins in the reaction mixtures can be directly analyzed by NMR, and (iv) application of novel stable isotope labeling schemes can facilitate resonance assignments [83, 84].…”

“…Moreover, expression of the membrane proteins in a membrane-free environment frequently results in rapid precipitation, necessitating subsequent refolding [44]. However by including carefully-selected detergents in the reaction mixture, cell-free systems have been shown to be capable of producing substantial amounts of folded membrane proteins [26, 27, 30, 33, 36, 44, 82]. Additional work has shown that inclusion of liposomes prepared from bacterial membranes and other bilayer systems may sometimes offer an alternative to micelles for membrane protein membrane insertion and folding [85, 86].…”

Limiting factors of the translation machinery

“…Bacteria were finally resuspended in two volumes of S30 buffer, containing 1 mM DTT, lysed by sonication (two bursts à 2-3 s) and centrifuged twice for 30 min at 30,000 × g and 4 • C (Beckmann rotor JA-25.50). The supernatant was adjusted to 400 mM NaCl using 4 M NaCl and incubated in a waterbath for 45 min at 42 • C. This preincubation method was adopted from Klammt et al (2007). Afterwards the lysate was dialysed against 2 l of S30 buffer containing 1 mM DTT using ZelluTrans dialysis membrane with a molecular weight cut off (MWCO) of 15,000 (Roth ® ).…”

Membrane protein production in Escherichia coli cell‐free lysates

“…Systematic screens of detergents or surfactants with defined working concentrations that are tolerated by the CF system have been published [3,65–69]. Most commonly used are long chain Brij derivatives as well as peptide surfactants, digitonin, the micelle forming short chain lipid di‐heptanoyl‐phosphocholine or fluorinated surfactants [16,61,69,70–74]. Suitable environments are still hard to predict.…”