In Vitro Production of Perdeuterated Proteins in H2O for Biomolecular NMR Studies

Abstract

The cell-free synthesis is an efficient strategy to produce in large scale protein samples for structural investigations. In vitro synthesis allows significant reduction of production time, simplification of purification steps and enables production of both soluble and membrane proteins.The cell-free reaction is an open system and can be performed in presence of many additives such as cofactors, inhibitors, redox systems, chaperones, detergents, lipids, nanodiscs and surfactants to allow the expression of toxic, membrane or intrinsically disordered proteins. In this chapter we present protocols to prepare E. coli S30 cellular extracts, T7 RNA polymerase and their use for in vitro protein expression. Optimizations of the protocol are presented for preparation of protein samples enriched in deuterium, a prerequisite for the study of high molecular weight proteins by NMR spectroscopy. An efficient production of perdeuterated proteins is achieved together with a full protonation of all the amide NMR probes, without suffering from residual protonation on aliphatic carbons. Application to the production of the 468 kDa TET2 protein assembly for NMR investigations is presented.

Determination of the two-component systems regulatory network reveals core and accessory regulations across Pseudomonas aeruginosa lineages

“…Cell-free expression was performed for all constructs after magnesium concentration optimization. The 55 RRs were expressed as previously described ( 46 ) in a final volume of 100 μl for each RR during 2 h at 23°C under gentle agitation, with a batch mode configuration. A total of 16 μg ml −1 of RR template DNA in pIVEX2.4d were added to a reaction mixture containing 1 mM of each of the 20 amino acids, 0.8 mM rNTPs (guanosine-, uridine-, and cytidine-5′-triphosphate ribonucleotides), 1.2 mM adenosine-5′-triphosphate, 55 mM HEPES, pH 7.5, 68 μM folinic acid, 0.64 mM cyclic adenosine monophosphate, 3.4 mM dithiothreitol (DTT), 27.5 mM ammonium acetate, 2 mM spermidine, 80 mM creatine phosphate, 208 mM potassium glutamate, 14 mM magnesium acetate, 250 μg ml −1 creatine kinase, 27 μg ml −1 T7 RNA polymerase, 0.175 μg ml −1 tRNA and 40% S30 E. coli bacterial extract.…”

G-Quadruplex Aptamer-Ligand Characterization

“…As seen in Figure 10 and Table S1 , complex 5’-FAM-AT11-L2-3´-TAMRA/PhenDC3 is able to localize NCL in UPCI-SCC-154 cells. The co-localization of the complex and NCL was determined by measuring the Manders’ coefficients M1 and M2 ( Table S1 ) [ 27 ]. These coefficients range from 0 to 1 and indicate the fraction of intensity in a channel that is located in pixels where there is above-zero intensity in the other color channel.…”

“…The cells were observed in a Zeiss LSM 710 confocal laser scanning microscope (Zeiss, Germany) coupled with a plane-apochromat, and a 63×/DIC M27 objective was used to capture the fluorescence images. After image acquisition, the degree of AT11-L2 with PhenDC3 colocalization with NCL was expressed in Manders coefficient values, which were calculated using the JaCop tool in the FIJI software [ 27 ]. Colocalization coefficients were calculated from four images and Costes’ automatic threshold was used.…”

Structural Perspective into the Interaction of an Oncogenesis‐Relevant pre‐miRNA G‐Quadruplex Ligand Carrier with the Protein Nucleolin

“…The sequence corresponding to NCL RNA binding domains 1 and 2 (NCL RBD1,2) was cloned into a pIVEX 2.4D vector. NCL RBD1,2 was synthesized in vitro using a cell‐free expression system [35] . Briefly, NCL RBD1,2 was expressed in a volume of 9 mL under RNAse‐free conditions in dialysis mode, with a 1/10 ratio of the reaction mixture to the feeding mixture for 16 h at 23 °C under gentle agitation.…”