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ExiProgen™ ProXpress PCR Template Kit allows production template DNA for protein expression quickly using PCR.
A two-step process is used for obtaining the template DNA. The detailed experimental workflow and the structure of the templates are as follows.
(A) Order primers for protein synthesis
Order primer sets for our ExiProgen™ ProXpress PCR Template Kit to receive primer sets for the first PCR for optimal protein expression within 48 hours.
If you want N-terminal 6x histidine tag, add N-8229 to your shopping cart; if you want C-terminal 6x histidine tag, add N-8230 to your shopping cart then eneter the desired sequences
1Design the forward primer to be 18 mer (not including the ATG codon) from the 5 'end of the target gene while do the reverse primer to be 18 mer (reverse sequence, stop codon not included) from the 3' end of the target gene as shown in the above example
2Select the position of 6x Histidine tag and then add [First primer F/R sets] with the appropriate position in the shopping cart; proceed to the payment afterwards
3Enter the sequence of forward primer and reverse primer in [Payment/Shipping Request].
4Wait until the ordered [First primer F/R sets] is supplied which will be manufactured by introducing 21 mer sequence at each 5 'end of the primer
(B) 1st PCR Reaction :
Primary target gene amplification using the ordered primer F/R sets and AccuPower® ProFi Taq PCR Premix included in the kit
(C) 2nd Overlapping PCR Reaction :
Template preparation for protein synthesis using the first PCR reaction as its template, with primer sets and cassette sets included in the kit
(D)Synthesized template DNA structures with ExiProgen™ ProXpress PCR Template Kit
▶ Experiment Results
1. Securing the Templates (2nd Overlapping PCR products)
Figure 1. Synthesis of linear template DNA with ExiProgen™ ProXpress PCR Template Kit
Each sample is loaded 100 ng on 1% TBE agarose gel. - M1; 100bp DNA Ladder(Bioneer, D-1030) - 1; SAV (0.6kb Template – pT) - 2; RNase H (0.7kb, Template – BL21(DE3) gDNA) - 3; hGH (0.8kb, Template – pT) - 4; CAT (0.9kb, Template - pBIVT) - 5; UDG (0.95kb, Template - BL21(DE3) gDNA) - 6; AcGFP (0.97kb, Template – pBIVT ) - 7; EVO (1kb, Template –pT) - 8; RFP (1kb, Template – pIVEX) - 9; Poly A polymerase (1.6kb, Template – pET15b) - M2; 1kb DNA Ladder(Bioneer, D-1040)
2. Synthesizing proteins using ExiProgen™ EC Protein Synthesis Kit
Figure 2. Synthesis of proteins with ExiProgen™
Each linear template DNA generated by ExiProgen™ ProXpress PCR Template Kit and those are used as template for protein synthesis with ExiProgen™ EC Protein Synthesis Kit. - M; AccuLadder™ Protein Size Marker (Low)(Bioneer, D-2020) - 1; SAV (13 kDa) - 2; RNase H (20 kDa) - 3; hGH (23 kDa) - 4; CAT (26.5 kDa) - 5; UDG (28 kDa) - 6; AcGFP (28 kDa) - 7; EVO (30 kDa) - 8; RFP (31kDa) - 9; Poly A polymerase (54 kDa)
Currently, the molecular weights (MW) of synthesizable proteins ranging from 10 kDa ~150 kDA were tested and validated. Though it is theoretically possible, it is not recommended to go over or under that scale.
Figure 1. SDS-PAGE data of various proteins synthesized from various templates M: AccuRapid™ Protein Size Marker (Broad) (Cat. No. D-2010, Bioneer) 1: CalmL3 (17.5 kDa) 2: DUSP3 (22 kDa) 3: CAT (24 kDa) 4: AcGFP (29 kDa) 5: ERFP (31 kDa) 6: EF-Ts (34 kDa) 7: VF (45 kDa) 8: BM3 (117 kDa)
The time it takes to obtain the proteins depends on the synthesized kit. For ExiProgen™ EC Protein synthesis Kit, it takes 6 hours for the expression to be completed. Refer to the table for more information on other kits. Additionally, after the final purification step, ExiProgen™ stores the target proteins in low temperature from 4°C to 8°C inside the reaction block. Even when the device is operated overnight, those can be also stably kept without separate refrigeration devices.
* It is recommended to use the Manual Kit for simpler controlling of expression volumes if only small scales of protein synthesis are required.
The template DNA should have the structure of T7 promoter, RBS, His-tag and T7 terminator as shown below. Depending on where the His-tag is attached, you can choose between two different types of expression, folding, and separation purification. At this time, the template type can be either plasmid DNA or PCR product. When preparing with a PCR product, it is advantageous to prepare several types of templates quickly, but the yield of protein synthesis may be slightly lower than that of plasmid DNA. Especially, when using SECF method for long time reaction for large-scale expression, the stability of template is important, so we recommend plasmid template.
1) When plasmid DNA is used as a template, It is recommended to use pBIVT vector (Cat. No. K-7350, Bioneer) which is exclusive to in vitro translation, but T7 system, RBS, and His-tag for purification can also be used. Using the AccuPrep® Nano-Plus Plasmid Mini Extraction Kit (Cat. No. K-3112, Bioneer), cloned plasmid DNA can be isolated with high-yield, high-purity only within 10 minutes
2) When using a PCR product as a template, ExiProgen™ ProXpress PCR Template Kit (Cat. No. K-7400, Bioneer) can be used for amplification of the target gene for preparing large quantities of the templates by PCR without the cloning step. For the best results, the purity of the acquired template DNA should be in the range of A260 / 280 ratio of 1.8 - 2.0.
Table. Related Products
Plasmid template generation by cloning
1) pBIVT Vector (Cat. No. K-7350)
2) pBT-7 vector (Gene synthesis service)
3) Other vector may also be used. (ex. pIVEX, pK7, pET vector series)
2) ExiProgen™ Protein Expression Optimization Kit (Cat. No. K-7410)
As the quantity of template DNA affects the efficiency of protein synthesis, it is very important to use an appropriate amount. Since the number of plasmid copies is important for the template DNA used, it should be quantitatively proportional to the size of DNA. In general, the amount of plasmid DNA added to 750 µl of expression volume is 1 µg/kb while the amount of linear DNA needed for the same expression volume is 500 ng (≤1 kb) or 1 µg (>1 kb). Please refer to the answer from Question 2) for more details.
If you want an optimal protein synthesis, we recommend an experiment that screens the amount of expressed protein according to the amount of template DNA used.
(Ex) An experiment using AccuRapid ™ Cell-Free Protein Expression Kit (Cat. No. K-7250) to optimize template DNA usage.
1. Prepare AccuRapid ™ Cell-Free Protein Expression Kit.
Expression volume: 45 µl
2. Prepare template DNA of the following concentrations (per 1 kb of plasmid DNA).
Standard DNA usage (1.34 µg/ml): 60 ng
DNA usage for tests: 120, 240, 360, 480, 600 ng
3. Proceed with the reaction referring to the manual of AccuRapid ™ Cell-Free Protein Expression Kit.
4. Verify the optimal amount of template DNA through SDS-PAGE analysis.
5. Get the optimal amount of DNA by converting from the expressed volume of the protein synthesis kit you are using (with the equation below).
Optimal DNA amount (ng) * expression volume of the kit you are using (µl) / 45 (µl)
Since the protein synthesis kit uses E. coli bacterial expression system, if the templates are optimized for E. coli codons, the protein expression levels and the success rates can be increased.
The purification methods used in ExiProgen™ utilize average diameter or 400 nm Silica Magnetic Nanobeads developed with Bioneer’s proprietary technology. On the surface of the beads, Ni-NTA is firmly immobilized, allowing purification of proteins by affinity purification methods through the strong binding of His-tag of the target proteins. These processes are fully automatic in ExiProgen™.
Due to the tertiary structure of the final product of proteins, if the His-tag at the N- or C-terminal is not exposed, to the surface, Ni-NTA can’t recognize it, lowering the purification efficiency. Thus, it is important to identify the protein structure and undergo a thorough check when designing the template DNA. If the protein structure is not known, it is advised test the expression and purification efficiency both templates with His-tag attached on N- or C- terminal. If those cannot be performed at both ends, it is recommended to increase the number of His-tag histidine to enhance them. If this method also does not work, then try attaching another protein which helps the protein expression to the N-terminal to produce a soluble fusion protein.
We are continuing to develop an efficient expression system, so please contact us if the protein expression fails.
Most of the time, this is caused by poor protein expression. It is recommended to use a manual kit AccuRapid™ to find the optimal protein expression condition on a small scale. Try running the same condition in ExiProgen™ for improved efficiency. If the expression level is high but the purification level is low, try changing the position of His-tag as explained in Question 7.
Each ExiProgen™ protein synthesis kit is consisted of two boxes: Kit ① and Kit ②.
It is highly recommended to store the purification kit (Kit ①) at 4℃ ~ 8℃ and Expression Kit (Kit ②) at -20℃ ~ -70℃. For the long-term storage, it is adviced to keep the E. coli extract included the Expression Kit (Kit ②) at -70℃.
For more details, please refer to the box and the user manual.
The cartridges is designed to be used up to 4 times. However, it is not recommended to use them overnight if you want to do so.
Store the them at the recommended temperature immediately after the ExiProgen™ operation. Conceal with cartridge covers and store cartridge ① at 4℃ to 8℃ and cartridge ② to -20 ° to -70 ° C.
The performance of the E. coli extract will be decreased on the occasion of repeated freezing and thawing. As it is packaged in a single tube for one-time use, please make sure to check the kit components and use it according to your experimental plans.