Principle of Protein Synthesis with ExiProgen™
This product is intended to be used with Bioneer's automated system ExiProgen™ for expression and purification of proteins, and the principle of the product is illustrated in the following figure.
Left panel: Manual method of protein expression/purification – sequential and tedious
Right panel: Bioneer's automated method of protein expression/purification – simply add DNA and press start!
Template DNA
The template DNA for expression by ExiProgen™ EC Protein Synthesis Kit must have a "T7 promoter
– ribosomal biding site (RBS) – target gene – T7 terminator" structure, and target gene must contain a histidine tag coding sequence at
N-terminus or C-terminus for the protein purification.
Maximization of Protein Expression
Protein can be normally synthesized with 6-10 µg of template DNA, but the yield of protein synthesized in the reaction depends on the amount of DNA used for expression.
Therefore, a preliminary experiment described as below is recommended to determine the amount of template DNA to maximize the expression of each protein using the AccuRapid™ Cell-free Protein Expression Kit.
Note that more DNA is not always better!
Example: Synthesis of poly(A) polymerase
1. Determination of the optimal amount of template DNA for the expression of poly(A) polymerase using AccuRapid™ Cell-Free Protein Expression Kit
; Poly(A) polymerase was maximally expressed with the AccuRapid™ kit when 400 ng of pET-poly(A) polymerase was used.
Lane M: AccuLadder™ Protein Size Marker (Broad), Lane NC: Negative Control, Lane PC: Positive Control (CAT), Lane 1 - 6: 50, 100, 200, 400, 600, and 800 (ng) of pET-poly(A) polymerase
2. Protein synthesis using ExiProgen™ EC Proteins Synthesis Kit
6.7 µg of pET-poly(A) polymerase was used to synthesize poly(A) polymerase with the EC1 kit (400 ng x 16.7 = 6.7 ug)
Lane M: AccuLadder™ Protein Size Marker (Low)
Lane E: Expression Sample
Lane P: Purification Sample
Kit Contents and Storage Condition
Kit-1
(Purification kit) |
Cartridge ① |
4 °C |
| Disposable filter tip |
Room temperature |
Kit-2
(Expression kit) |
Cartridge ② |
-20 °C |
| Positive control DNA in 1.5 ml tube |
-20 °C |
| E. coli cell extract in 8-strip tubes |
-70°C |
| Elution tube and cap |
Room temperature |
This product contains a "Purification Kit (Kit 1)" and an "Expression Kit (Kit 2)".
A Cell Extract (8-well strip tube) containing
T7 RNA Polymerase and ribosomes is included separately.
Cartridge ② contains a master-mix containing reagents necessary
for RNA synthesis and protein expression, and Cartridge
① contains Ni-NTA magnetic beads and other buffers necessary for purification of His-tagged proteins.
(The Ni-NTA magnetic beads are provided in a 20% ethanol at 10% (w/v) concentration.)
Experimental Data
1. Reproducibility: ExiProgen™ EC Protein Synthesis Kits provide reproducible results, the first time and every time.
Figure 1. Expression and purification of CAT. Reproducibility is seen in all 16 wells with no detectable variation between wells.
Lane 1~16: Number of the well,
M : AccuLadder™ Protein Size Marker (Low),
E : Expression sample,
P : Purification sample
2. Flexibility: Proteins as small as 10 kD and as large as 120 kD have been expressed/purified with ExiProgen™.
Figure 2. Expression and purification of various proteins. Up to 16 types of proteins can be expressed and purified simultaneously with an average of over 90% purity.
Top of the lane: Name of the protein,
M : AccuLadder™ Protein Size Marker (Low),
E : Expression sample,
P : Purification sample
3. Control: Each kit comes with a Green Fluorescent Protein positive control providing confidence in the results.
Figure 3. Detection of fluorescence emitted from fluorescent proteins synthesized with ExiProgen™.
This result indicates that these synthesized SfGFP, RFP, and AcGFP are functionally active.
Left panel: Color of each protein elution samples detected with naked eyes.
Right panel: Fluorescence from protein elution samples detected with UV illuminator.
Figure 4. Expression and purification of fluorescent proteins.
Top of the lane: Protein name,
Lane M: AccuLadder™ Protein Size Marker (Broad),
Lane E: Expression sample,
Lane P: Purification sample
4. Functionality: Proteins expressed/purified with ExiProgen™ can be assayed for functionality and the resulting proteins will generally yield up to 100 µg or > 90% pure protein.
Many established recombinant proteins can be expressed in E. coli extracts with full functional activity.
Figure 5. Expression and purification of DUSP3 (protein tyrosine phosphatase).
Lane M: AccuLadder™ Protein Size Marker (Low),
Lane E: Expression sample,
Lane P: Purification sample
Figure 4. DUSP3 synthesized with ExiProgen™ has an enzyme activity.
TDUSP3 phosphatase activity was measured by incubating with 500 uM 3-O-methylfluorescein phosphate (OMFP).
(Reaction buffer : 100 mM Tris-HCl (pH8.2), 40 mM NaCl, 1 mM DTT, 20% Glycerol)