EF-P

Other proteins

EF-P

PFS052-0.5-EX

Size Price (USD)
500 µL reaction $44.00

PFS052-10-EX

Size Price (USD)
2 mL x 5 reaction $700.00

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CONTENTS

Overview

“EF-P” is a supplement by adding to PUREfrex®, 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.

Experiment Workflow

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.


Protocols

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.

  1. Thaw Solution I, Cysteine and GSH by incubation at room temperature or 37 °C for 1 minute completely dissolving, and then cool on ice.

  2. Thaw Solution II, III and EF-P on ice.

  3. Mix each solution by vortex and centrifuge briefly to collect each solution at the bottom.

  4. Assemble the reaction mixture in a tube as follows. Add the template DNA to 1 - 3 ng/µL per 1 kb.



Water 6.5-X μL
Solution I *1 8 μL
10 mM Cysteine
1 μL
80 mM GSH 1 μL
Solution II 1 μL
Solution III 2 μL
40 μM EF-P
0.5 μL
Template DNA *2 X μL
Total 20 μL

  1. Incubate the tube at 37 °C for 2 - 6 hours with heat block or water bath.

  2. Analyze the synthesized products. Please add the same amount of H2O to the reaction for the sample of SDS-PAGE.

  1. This volume is different from Solution I of PUREfrex®2.0.
  2. Please visit the template DNA preparation site.

Specifications

Kit components   <EF-P>



Reagent Quantity
for 500 μL rxn
Quantity
for 2 mL x 5 rxn
Description Storage *1
EF-P 12.5 µL 50 μL x 5 EF-P with no tags (40 µM) -80 °C *2
Dilution buffer
500 µL 500 μL x 1 30% glycerol buffer
-20 °C
  1. Indicated temperature shows storage temperature after opening the kit. Please store the kit at -80 °C before opening.
  2. 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.

Datasheet


PFS052-0.5-EX

Downloads

PFS052-10-EX

Downloads

SDS



Note

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"

Applications


On the Resources page, you can find posters and technical notes about PUREfrex®. Additionally, there are papers that use PUREfrex® or PURE system technology.


Related Products

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


PUREfrex®1.0

for Basic Research and Customized kit

Catalog No.

  • PF001-0.25-EX, PF001-0.25-5-EX
more

PUREfrex®2.0

for Syntesis Proteins

Catalog No.

  • PF201-0.25-EX, PF201-0.25-5-EX
more

PUREfrex®2.1

for Forming Disulfide Bonds

Catalog No.

  • PF213-0.25-EX, PF213-0.25-5-EX
more

Supplements – Chaperones –

These are supplement for PUREfrex® which is molecular chaperone to be aggregate-prone protein in a soluble form.


DnaK Mix

HSP70

Catalog No.

  • PF003-0.5-EX
more

GroE Mix

HSP60

Catalog No.

  • PF004-0.5-EX
more

Supplements – Forming disulfide bonds –

This is supplement for synthesizing proteins containing disulfide bonds in an active form.


DsbC Set

E.coli isomerase, GSSG

Catalog No.

  • PF005-0.5-EX
more

PDI Set

Human isomerase, Ero1α, GSSG

Catalog No.

  • PF006-0.5-EX
more

FAQ

Can I synthesize eukaryotic proteins using the PUREfrex® kit?
YES.
The PUREfrex® is a reconstituted cell-free protein synthesis kit composed of E. coli ribosomes and translation factors, but eukaryotic proteins from such as mammalian and plant can also be synthesized. The productivity of the target protein may depend on the property of nucleotide sequence encoding the target protein, such as GC contents or frequency of rare codon.
How much can I synthesize the target protein using the PUREfrex® kit?
It depends on the target protein.
Dihydrofolate reductase from E.coli can be synthesized with about 600 μg per mL of the reaction.
Can I synthesize proteins of more than 100 kDa?
YES.
We have synthesized the protein of about 120 kDa using PUREfrex®.
What do you recommend the reaction condition for PUREfrex®?
We recommend that the protein synthesis using PUREfrex® is carried out at 37 °C for 2-6 hours.
Can I synthesize and purify a tagged protein?
YES.
You can use any tag, because all of protein components of PUREfrex® have no tag for purification or detection. For example, the target protein with histidine tag can be purified with metal-chelating resin by applying reaction mixture directly, after synthesizing.
Is synthesized protein modified by glycosylation or phosphorylation?
NO.
There is no post-translational modification, because PUREfrex® is constituted by translation factors only.
Does PUREfrex® contain any molecular chaperones?
NO.
PUREfrex® does not contain any chaperones, but you can add chaperones, such as Hsp70.
GeneFrontier has been developing molecular chaperones suitable for PUREfrex® kit, DnaK Mix (#PF003-0.5-EX) and GroE Mix (#PF004-0.5-Ex).
Can I synthesize a protein containing disulfide bonds with PUREfrex®?
DsbC Set (#PF005-0.5-EX) and PDI Set (#PF006-05-EX) are supplements by adding to PUREfrex® for synthesizing proteins containing a disulfide bond in an active form. PUREfrex® 2.1 (#PF213-0.25-EX) is recommended to optimize the reaction conditions.
Can I synthesize a membrane protein with PUREfrex®?
YES.
In the most of the cases, a synthesized membrane protein may form aggregates. To obtain a membrane protein inserted into a lipid bilayer, you may add a lipid bilayer such as liposome to PUREfrex® when you synthesize the membrane protein.
Can I synthesize a radiolabeled protein with [35S] methionine or [3H] leucine?
YES.
A radiolabeled protein can be synthesized by adding radioisotope-labeled amino acids, such as [35S] methionine or [3H] leucine, to PUREfrex®. The amino acid content is 0.5 mM for PUREfrex®1.0 and 2 mM PUREfrex®2.0. Please consider that point and optimize conditions for your labeling.
Can I use any promoter besides T7 promoter?
We recommend the template DNA with T7 promoter, because PUREfrex® contains T7 RNA polymerase for transcription. When you use other polymerases, please generate template DNA with the suitable promoter for the selected polymerase.
I can’t get DHFR using DHFR DNA in the kit as positive control.
1. The kit may be inactivated for some reasons. To avoid the inactivation, please store the kit at suitable temperature and divide into aliquots to reduce freeze-thaw cycles as much as possible.

2. The kit may be contaminated by nuclease. To avoid a nuclease contamination, please use nuclease-free water, reagents and materials.
I can get DHFR using DHFR DNA in the kit. But I can’t get my protein of interest or can get it with very low amount.
1. The kit may be inactivated for some reasons. To avoid the inactivation, please store the kit at suitable temperature and divide into aliquots to reduce freeze-thaw cycles as much as possible.

2. The kit may be contaminated by nuclease. To avoid a nuclease contamination, please use nuclease-free water, reagents and materials.
Sometimes the plasmid is contaminated with RNase through a plasmid preparation process. So, please confirm that RNase is removed from the plasmid.

3. The construct of template DNA may be incorrect. It is necessary to contain the sequence of T7 promoter, ribosome binding site, initiation codon, and stop codon for the template DNA for PUREfrex®.

4. Secondary structure of transcripts may prevent the translation reaction. In this case, please optimize the template sequence to solve the problem of secondary structure.