In vitro translation-transcription reactions were performed using the Purexpress In Vitro Protein Synthesis system (NEB #E6800). The reaction solution was prepared in the following order: 10 μL solution A (NEB #E6800), 7.5 μL solution B (NEB #E6800), 2 μL E. coli RNA polymerase (NEB #M0551), 1 μL RNase inhibitor, 500 ng reduced, H2Sn-treated or H2O2-treated protein, 200 ng DNA fragment containing PtrxC-mKate, and RNase free water. The total volume was 25 μL. The solution was incubated at 37 °C for 3 h. After reaction, the translated mKate was diluted four times with distilled water, and assayed by using the Synergy H1 microplate reader. The excitation wavelength was set to 588 nm, and the emission wavelength was set to 633 nm. The fluorescence intensity from reduced OxyR was used as standard; fluorescence intensities from other groups were divided by the standard to calculate the relative expression levels.
Purexpress in vitro protein synthesis system
Manufactured by New England Biolabs
Sourced in United States
The PURExpress in vitro protein synthesis system is a cell-free protein expression kit developed by New England Biolabs. It enables the production of proteins from DNA or mRNA templates without the need for live cells. The system provides the necessary components, including ribosomes, tRNAs, and enzymes, to facilitate in vitro protein synthesis.
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Lab products found in correlation
Soybean l α phosphatidylcholine, by Merck Group (2 mentions)
E coli rna polymerase, by New England Biolabs (1 mentions)
Mini protean tetra system chamber, by Bio-Rad (1 mentions)
Roti blue quick solution, by Carl Roth (1 mentions)
Mini protean tris tricine gel, by Bio-Rad (1 mentions)
Tnt t7 insect cell extract protein expression system, by Promega (1 mentions)
Purexpress δ ribosome kit, by New England Biolabs (1 mentions)
9 protocols using purexpress in vitro protein synthesis system
1
In Vitro Protein Expression Assay
2
In vitro Protein Synthesis and Pull-down Assay
3
Cell-Free Synthetic Biology System
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IVT2H reagents are described previously20 (link)21 (link)22 . Briefly, IVT2H contained 144 nM purified E. coli RNA polymerase core enzyme, 1.2 μM purified recombinant E. coli IHF, 0.8 units/μl murine RNase inhibitor, the PURExpress®in vitro protein synthesis system (New England Biolabs), 0.2 ng/μl (45 pM) plasmid DNA expressing σ54, 4.4 nM linear reporter DNA expressing GFP and 0.2 ng/μl (60 pM) plasmid DNA expressing hybrid fusion protein AD-MDM2, in which the activation domain (AD, residues 1–296) of PspF was fused to the full-length human MDM2 protein. The genes for the wild-type p53 peptide (p53p, residues 17–26: ETFSDLWKLLPE) and the peptide inhibitor (PMI: TSFAEYWNLLSP) were synthesized and fused to the N-terminus of the DNA binding domain Cro (DB). The linear DNA constructs expressing p53p-DB, PMI-DB or the PMI library-DB were used in drop-based microfluidics experiments.
4
Cell-free Protein Synthesis and Pull-down Assay
5
In vitro Degradation Assay for Bacterial Proteins
6
In Vitro Protein Interaction Assay
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Pull-down assay was performed with proteins produced using an in vitro transcription/translation system as described (Lee et al. 2013 (link)) with some modifications. Proteins were produced using the cell-free PURExpress in vitro protein synthesis system (New England Biolabs) for 3 h at 37°C. DNA templates were prepared according to the manufacturer's instructions. To synthesize the hspQ-Flag, clpS-HA, and rssB-HA genes, we used primers 16936/16937 for hspQ-Flag, 15949/16938 for clpS-HA, and 16939/16940 for rssB-HA. At the end of the reaction, samples were diluted with 20 vol in 1× TBS buffer. Diluted reactions were mixed in 500 µL of 1× TBS and incubated for 2 h at room temperature. Next, samples were pulled down with either anti-HA, anti-His, or anti-Flag antibodies for 2 h at room temperature. Pulled-down samples were analyzed by Western blotting using anti-HA, anti-His or anti-Flag antibodies.
7
Comparing Cell-Free Protein Expression Systems
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We tested three different commercially available cell-free expression systems: the PURExpress In Vitro Protein Synthesis System (New England Biolabs, Ipswich, MA, USA), the S30 Extract System (Promega, Madison, WI, USA), and the TnT T7 Insect Cell Extract Protein Expression System (Promega) based on the S. frugiperda cell line Sf21. We followed the manufacturer’s recommendations for each kit, using 1.5 mL Eppendorf tubes for each reaction. The S30 Extract System lacks methionine, so we added this amino acid to match the concentrations in the other systems. All tubes were stored at −20 °C after the reaction. Protein synthesis was confirmed by 1D SDS-PAGE. We mixed 1 µL of the reaction with 1 µL of Tricine sample buffer and incubated it for 5 min at 95 °C. The sample was then loaded onto a 16.5% Mini-PROTEAN Tris-Tricine Gel (Bio-Rad, Hercules, CA, USA) and placed in a Mini-PROTEAN Tetra System chamber (Bio-Rad) filled with 10x Tris/Tricine/SDS running buffer. After electrophoresis at 100 V for 100 min, the gel was stained overnight with Roti-Blue quick solution (Carl Roth, Karlsruhe, Germany).
8
Toe-Printing Analysis for Protein Synthesis
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Toe-printing analysis was performed using the RST2 template (49 (link)) as previously described (50 (link)). Cell free translation reactions, carried out in the PURExpress in vitro protein synthesis system (New England Biolabs, Ipswich, MA, USA) were supplemented with 50 μM thiostrepton or 100 μM of peptides which were added in water and samples (5 μl volume) were incubated for 15 min at 37°C prior to the primer extension phase of the procedure. The control reaction had no inhibitor. Primer extension was carried out for 15 min, after which the samples were processed as described (50 (link)).
9
In Vitro Protein Synthesis Assay
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Translation was assayed in the PURExpress In Vitro Protein Synthesis system (E6800S, New England BioLabs) in 10-µL reaction volumes. Detection of FLAG-tagged TisB (in Fig. 2B ) or by radioactive labeling (in Fig. 5C ) is described in SI Appendix, SI Materials and Methods . To test 70SΔS1 functionality, the PURExpress ΔRibosome Kit (E3313S, New England BioLabs) was used instead and supplemented with purified 30S and 50S subunits.
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