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“EF-P” is a supplement by adding to PUREfrex®series, cell-free protein synthesis kits.
EF-P (elongation factor P) is one of translation factors in E. coli and a homolog of eukaryotic initiation factor 5A (eIF5A). EF-P has a promoting activity for the peptide bond formation between the first methionine and the second amino acid and the elongation of consecutive proline residues (3 or more prolines).
Lysine at 34th of EF-P is post-translationally modified and the modification is important for the activity. “EF-P” kit includes post-translationally modified EF-P from E.coli.
Lysine at 34th of EF-P is post-translationally modified to β-lysllysine by EpmA (YjeA/GenX) and EpmB (YjeK) and the modification is important for the activity.
MS1 Extracted Ion Chromatogram of the isolated recombinant EF-P from E.coli.
General procedures for using PUREfrex® to synthesize functional proteins are shown in Figure.
Start by preparing the template DNA suitable to the protein synthesis with PUREfrex® and examine whether the protein of interest is synthesized. Redesign the nucleotide sequence of the template DNA if too little protein is synthesized.
Add EF-P as supplement to PUREfrex® reaction mixture if the amout of synthesized protein is low and it contains consecutive proline residues.
This is a standard protocol for synthesizing proteins using EF-P and PUREfrex®2.1. For example, please assemble 20 µL of reaction mixture as below, in which the final concentration of each reagent is 0.5 mM Cysteine, 4 mM GSH and 1 µM EF-P.
- Thaw Solution I, Cysteine and GSH by incubation at room temperature or 37 °C for 1 minute completely dissolving, and then cool on ice.
- Thaw Solution II, III and EF-P on ice.
- Mix each solution by vortex and centrifuge briefly to collect each solution at the bottom.
- Assemble the reaction mixture in a tube as follows. Add the template DNA to 1 - 3 ng/µL per 1 kb.
|Solution I *1||8 μL|
|10 mM Cysteine
|80 mM GSH||1 μL|
|Solution II||1 μL|
|Solution III||2 μL|
|40 μM EF-P
|Template DNA *2||X μL|
- Incubate the tube at 37 °C for 2 - 6 hours with heat block or water bath.
- Analyze the synthesized products. Please add the same amount of H2O to the reaction for the sample of SDS-PAGE.
- This volume is different from Solution I of PUREfrex®2.0.
- Please visit the template DNA preparation site.
Kit components <EF-P>
for 500 μL rxn
for 2 mL x 5 rxn
|EF-P||12.5 µL||50 μL x 5||EF-P with no tags (40 µM)||-80 °C *2|
||500 µL||500 μL x 1||30% glycerol buffer
- Indicated temperature shows storage temperature after opening the kit. Please store the kit at -80 °C before opening.
- For storage at -80 °C, the rest of solution should be frozen rapidly in liquid nitrogen or dry ice/ethanol. Please divide into aliquots, if necessary, and avoid refreeze and thaw as much as possible.
EF-P is developed for in vitro research only. EF-P should not be used for the therapy, diagnostic or administration to animals including human and should not be used as food or cosmetics etc. To avoid the contamination of nuclease, nuclease-free-treated water, reagents and materials should be used. We also recommend wearing gloves and mask.
"PUREfrex® is Registered in U.S. Patent and Trademark Office"
On the Resources page, you can find posters and technical notes about PUREfrex®.
Additionally, there are papers that use PUREfrex® or PURE system technology.
When your protein needs an assistance of molecular chaperone or a formation of a disulfide bond to form a correct conformation, we have supplemental reagents by simply adding to PUREfrex® series.
Cell-free Protein Synthesis Kits
Supplements – Chaperones –
These are supplement for PUREfrex® which is molecular chaperone to be aggregate-prone protein in a soluble form.
Supplements – Forming disulfide bonds –
This is supplement for synthesizing proteins containing disulfide bonds in an active form.