The iSAT reaction was performed as described previously (16 (link),17 (link)) with slight modifications on the protein concentration and the operon of plasmid. Briefly, the iSAT reaction was performed in 57 mM Hepes-KOH, 1.5 mM spermidine, 1 mM putrescine, 10 mM Mg(Glu)2, and 150 mM KGlu at pH 7.5 with 2 mM DTT, 0.33 mM NAD, 0.27 mM CoA, 4 mM sodium oxalate, 2% w/v PEG-6000, 2 mM amino acids (Sigma-Aldrich), 1 nM pY71sfGFP plasmid encoding superfolder GFP (M. Jewett), 0.1 mg/mL T7 RNA polymerase, 42 mM phosphoenolpyruvate (Roche) and NTP+ mix (1.6 mM ATP (Sigma), 1.15 mM of GTP, CTP and UTP each (Sigma), 45.3 μg/μL tRNA from E. coli MRE 600 (Roche), 227.5 μg/μL Folinic acid. The above components were premixed. A 5.36 μL aliquot of the premix was pipetted into 5 μl of S150 extract. Ribosomal proteins and plasmid encoding the engineered rrnB operon were added to a final concentration of 0.6 μM and 4 nM respectively. iSAT reactions of 15 μL each were performed in 96 well plates (Applied Biosystems) and incubated in a CFX Connect Real-time System (BIORAD) at 37 °C for variable time. sfGFP production was detected by fluorescence measurement at 5 min intervals (excitation: 450–490 nm, emission: 510–530 nm).
Phosphoenolpyruvate
Manufactured by Roche
Sourced in United States, Germany
Phosphoenolpyruvate is a key intermediate in the glycolytic pathway, serving as a high-energy phosphate donor for the conversion of ADP to ATP. It is an important substrate in various biochemical processes, such as gluconeogenesis and the citric acid cycle.
Automatically generated - may contain errors
Lab products found in correlation
Pyruvate kinase, by Roche (3 mentions)
Lactate dehydrogenase, by Roche (3 mentions)
Adenosine triphosphate (atp), by Roche (3 mentions)
Amino acid, by Merck Group (2 mentions)
T7 rna polymerase, by Roche (2 mentions)
Trna from e coli mre 600, by Roche (2 mentions)
96 well plate, by Thermo Fisher Scientific (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Nadph, by Roche (2 mentions)
Cfx connect real time system, by Bio-Rad (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Fp 8500, by Jasco (1 mentions)
Peg6000, by Roche (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Choline, by Merck Group (1 mentions)
Pyruvate kinase, by Merck Group (1 mentions)
Restriction endonuclease, by New England Biolabs (1 mentions)
Dntps, by Promega (1 mentions)
Sds page gel, by GenScript (1 mentions)
12 protocols using phosphoenolpyruvate
1
In Vitro Cell-Free Protein Synthesis
2
Biotinylation and Protein Assay Protocol
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The following chemicals were purchased from Sigma-Aldrich (St Louis, MO): ammonium chloride (Cat #A9434), phosphoenol-pyruvate (Cat#10108294001, Roche), pyruvate kinase/lactic dehydrogenase (PK/LDH; Cat#P0294), NADPH (Cat#NADPH-RO), ouabain (Cat#O3125) and concanamycin A (Cat#C9705). B27 supplement (Cat#17504-044) and glutaMAX (Cat#35050) were purchased from Invitrogen. N-[6-(biotinamido) hexyl]-3′-(2′-pyridyldithio) propionamide-biotin (Cat#21341) was from Thermo Scientific, Rockford, IL.
3
Fluorescence Titration of ClpB NBD1-M Variants
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Fluorescence titrations were performed at 25 °C in buffer A using a JASCO FP-8500 fluorescence spectrometer (JASCO Germany GmbH) as described previously (25 (link)). The excitation wavelength was set to 296 nm to facilitate selective excitation of protein-bound MANT-dADP via FRET from nearby tryptophan residues. The MANT fluorescence signal was monitored at 441 nm. Direct titrations of ClpB NBD1-M variants (at 2 or 20 μm ) with MANT-dADP (2–50 μm ) were used to determine the binding affinity of MANT-dADP, which was subsequently applied as the reference KD in displacement titrations to determine KD(ADP) or KD(ATP). Here, ClpB NBD1-M variants (at 2 or 20 μm ) were incubated with MANT-dADP (15–40 μm ) and subsequently titrated with ADP (2.5–300 μm ) or ATP (125–20,000 μm ). ATP titrations were performed in the presence of 2 mm phosphoenolpyruvate and 0.01 mg/ml pyruvate kinase (Roche Applied Science) as an ATP-regenerating system. The data were corrected for dilution effects and analyzed with a cubic equation for competing ligands using the initial concentrations of protein and MANT-dADP as well as the KD(MANT-dADP) from the direct titration as input values (31 (link)). The program GraFit version 5.0 was used for data fitting.
4
Cell-free iSAT protein expression
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The iSAT reaction was performed as described previously (21 (link),22 (link)) with slight modifications. Briefly, the iSAT reaction was performed in 57 mM HEPES–KOH, 1.5 mM spermidine, 1 mM putrescine, 10 mM Mg(Glu)2, and 150 mM KGlu at pH 7.5 with 2 mM DTT, 0.33 mM NAD, 0.27 mM CoA, 4 mM oxalic acid, 2% w/v PEG-6000, 2 mM amino acids (Roche), 1 nM pY71sfGFP plasmid encoding superfolder GFP (M. Jewett), 0.1 mg/ml T7 RNA polymerase, 42 mM phosphoenolpyruvate (Roche) and NTP + mix (1.6 mM ATP (Sigma), 1.15 mM of GTP, CTP and UTP each (Sigma), 45.3 μg/μl tRNA from E. coli MRE 600 (Roche), 227.5 μg/μl Folinic acid pH 7.2). The above components were premixed. The final concentration with respect to the total volume of the iSAT reaction is provided. A 5.6 μl aliquot of the premix was pipetted into 6 μl of S150 extract. Ribosomal proteins and plasmid encoding the rrnB operon (pT7rrnb; provided by M. Jewett) were added to a final concentration of 0.4 and 4 nM, respectively. iSAT reactions of 15 μl each were performed in 96-well plates (Applied Biosystems) and incubated in a StepOnePlus Real-time PCR System (Applied Biosystems) at 37°C for variable time. sfGFP production was detected by fluorescence measurement at 5 min intervals (excitation: 450–490 nm, emission: 510–530 nm).
5
Molecular Cloning and Protein Purification
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All chemicals were reagent or molecular biology grade. Platinum Pfu polymerase and DNA size markers were from ThermoScientific. dNTPs were from Promega. Restriction endonucleases and Sticky-End Master Mix Ligase were from New England Biolabs. Agarose gel purification, PCR product clean-up, and plasmid mini prep columns were all products of Qiagen. Human liver cDNA library was a product of ResGen (Invitrogen). All oligonucleotides were supplied by IBA GmbH, Göttingen, Germany. Choline and pyruvate kinase were from Sigma. ATP, NADH, phosphoenolpyruvate, and lactate dehydrogenase were purchased from Roche. SDS-PAGE gels were purchased from GenScript, and run using the supplied MOPS buffer. CM5 sensor chip for SPR was from GE Healthcare Life Sciences and used on a Biacore T200 system.
6
ATPase Activity Determination Methods
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The ATPase activities of proteins obtained after various purification procedures were determined by NADH-coupled spectrophotometric assays69 (link). 10 μg of DM-purified or nanodisc-reconstituted protein was added to 140 μL of reaction buffer containing 50 mM HEPES–KOH pH 8.0, 60 μg/mL pyruvate kinase (Roche), 16 μg/mL lactate dehydrogenase (Roche), 10 mM phosphoenolpyruvate (Roche), 0.3 mM NADH, 3 mM ATP, 10 mM MgCl2, and 0.17% (w/v) DM in the presence or absence of 250 μM peptide. For proteins reconstituted into nanodiscs, DM was not added to the reaction buffer. For lipid-stimulated ATPase assays, the thin film of lipids was hydrated in 100 μL of buffer containing 50 mM HEPES–KOH pH 8.0 and 10 mM MgCl2 to yield a 10 mM solution. After sonication of the lipid solution for 10 min to decrease its turbidity, the reaction was initiated by adding varying concentrations of lipids to the mixture. ATP hydrolysis activity was monitored at 37 °C for 3 min by measuring the rate of decline of NADH absorbance at 340 nm. To inhibit ATP hydrolysis, the reaction mixture was incubated with 3 mM AMP-PNP or 8 mM NaF/2 mM BeSO4 for 5 min at RT. The kinetic parameters of ATP hydrolysis were calculated by fitting the initial rates of ATP hydrolysis to the Michaelis–Menten equation using PRISM 4.0 (GraphPad software).
7
E. coli CFPS of GRFT Optimization
8
Cell-Free Protein Synthesis with Engineered Enzymes
9
Comprehensive Fluorescent Probe Preparation
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Pluronic L121, Pluronic L61, glycerol, sucrose, KCl, MgCl2, CaCl2, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES > 99.5%), 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS > 96%), carboxyfluorescein, sulforhodamine B, rhodamine 6 G, eriochrome black T, polyethylene glycol (PEG, MW 6000 Da), poly(vinyl alcohol) (PVA, MW 13000–23000 Da, 87–89% hydrolyzed), poly(allylamine hydrochloride) (MW 17500 Da), FITC, NHS-Fluorescein, ATP disodium salt, ADP sodium salt, valinomycin (>98%), pyruvate kinase from rabbit muscle (Type III, 350–600 units mg−1 protein), lactic dehydrogenase from rabbit muscle (Type XI, 600-1,200 units mg−1 protein), Bovine Serum Albumin (BSA, Product No. A7030), Bradford Reagent (Product No. B6916), NADH (reduced disodium salt, Grade II), DL-dithiothreitol (DTT > 98.0% for molecular biology), chloroform, cyclohexane, n-octadecyltrimethoxysilane, HCl (36.5–38.0%), NaOH (>98.0%, ACS reagent) were purchased from Sigma Aldrich. 2-[methoxy(polyethyleneoxy)propyl]trimethoxy silane was purchased from Gelest. Phosphoenolpyruvate was purchased from Roche. Actin (>99%, rabbit muscle) was purchased from Cytoskeleton. Pluronic F-68 (Poloxamer P188), Atto 633 NHS ester, Atto 590, and Alexa Fluor 488 conjugated phalloidin were purchased from ThermoFisher. Poly(butadiene)-b-poly(ethylene glycol) (PB-PEO) was purchased from PolymerScience. (Canada).
10
Characterization of Membrane Transport
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Dioleoyl phosphatidyl choline was purchased from Avanti Polar Lipids, Alabaster, Alabama. Phosphoenolpyruvate, pyruvate kinase, lactate dehydrogenase, NADH, and ATP (disodium salt, special quality) were supplied from Roche Life Science, and apyrase VI and ouabain from Sigma-Aldrich. Oxonol VI, 5-iodoacetamidofluorescein (5-IAF), RH237, RH421, and P3-1-(2-nitro)phenylethyladenosine-5′-triphosphate (caged ATP) were purchased from Molecular Probes (Eugene, Oregon). Na+ and K+ salts were used in Suprapur quality (Merck). All other reagents were obtained at the highest quality available.
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