Principle of Cell-Free Protein expression system
Template DNA
The template DNA for expression by AccuRapid™ Cell-Free Protein Expression 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.
Note 1) We recommend cloning the template DNA into our in vitro transcription/translation optimized pBIVT vector set (sold separately).
Note 2) Sometimes pET vector series such as pET15b, 22b, 23a, and 28b can also be used for the kit, though this should be tested. In addition PCR products containing T7promoter, ribosomal binding site, T7 terminator, and His-tag encoding sequences at N- or C-terminus can be used for proteins synthesis as a template DNA.
The ExiProgen™ ProXpress PCR Template Kit can generate PCR templates for this purpose.
Note 3) It is recommended to use E.coli codon-optimized gene constructs, which will improve protein expression.
Note 4) The DNA template for protein expression can be synthesized through our Gene Synthesis Service, which optimizes the codons for E.coli protein expression.
The synthesized gene can be cloned into our in vitro transcription/translation vector by request. Please refer to our homepage (www.bioneer.com) for additional information.
Kit Contents
| AccuRapid™ Master mix |
170㎕×3 tube(Red Cap) |
| AccuRapid™ E.coli extract |
100㎕×3 tube(Blue Cap) |
| DNA template |
24㎕×1 tube(Green Cap) |
| DEPC-DW |
1㎖×1 tube(White Cap) |
Storage conditions
-20°C (E.coli extract ; -70°C)
Experimental Data
1. Protein Expression using AccuRapid™ Cell-Free Protein Expression Kit
Figure 1. Protein expression Data of AccuRapid™ Cell-Free Protein Expression kit (SDS-PAGE and Coomassie Brilliant Blue staining)
Lane M; AccuLadder™ Protein Size Marker (Low),
Lane 1,3,5; Negative control (No-DNA),
Lane 2; CAT (Chloramphenicol acetyl transferase),
Lane 4; GFP (Green fluorescence protein),
Lane 6; PTP1B(Protein tyrosine phosphatase 1B)
2. AccuRapid™ Cell-Free Protein Expression Kit expresses proteins efficiently
Figure 2. Comparison of protein expression between AccuRapid™ Cell-Free Protein Expression Kit and protein
expression kits from other companies (SDS-PAGE and Coomassie Brilliant Blue staining).
Lane M; AccuLadder™ Protein Size Marker (Low),
Lane 1,3,5 ; Negative control (No-DNA),
Lane 2; Bioneer's Kit and control DNA,
Lane 4; Company A's Kit and control DNA,
Lane 6; Company B's Kit and control DNA
3. Determination of the optimal amount of template DNA for cell-free protein synthesis using AccuRapid™ Cell-Free Protein Expression Kit
3.1. pBIVT vector construct: GFP
Figure 3. Determination of the optimal amount of template DNA for the expression of GFP using AccuRapid™ Cell-
Free Protein Expression Kit . GFP was maximally expressed with the AccuRapid™ Cell-Free Protein Expression Kit when 200 ng of pBIVT-GFP was used.
Lane M; AccuLadder™ Protein Size Marker (Low),
Lane 1; Negative Control (No-DNA),
Lane 2; CAT (Chloramphenicol acetyl transferase),
Lane 3 ~9; pBIVT-GFP (50, 100, 200, 400, 600, 800, 1000 (ng))
3.2. pET vector construct: MMLV RTase
Figure 4. Determination of the optimal amount of template DNA for the expression of MMLV RTase using AccuRapid™ Cell-Free Protein Expression Kit.
MMLV RTase was maximally expressed with the AccuRapid™ Cell-Free Protein Expression Kit when 600 ng of pET22b-MMLV RTase was used.
Lane M; AccuLadder™ Protein Size Marker (Broad),
Lane 1; Negative Control (No-DNA),
Lane 2; CAT (Chloramphenicol acetyl transferase),
Lane 3 ~9; pET22b-MMLV RTase (50, 100, 200, 400, 600, 800, 1000 (ng))
AccuRapid™ Cell-Free Protein Expression Kit's positive control DNA expression test
Figure 3. AccuRapid™ Cell-Free Protein Expression Kit's Positive control DNA expression test (SDS-PAGE and Coomassie Brilliant Blue staining)
Lane M; AccuLadder™ Protein Size Marker (Low)
Lane 1,3,5,7 ; Negative control (No-DNA)
Lane 2; Bionee's Kit
Lane 4; Company A's kit
Lane 6; Company B's kit
Lane 8; Company C's kit
Company A's kit positive control DNA expression test
Figure 4. Company A's kit positive control DNA expression test (SDS-PAGE and Coomassie Brilliant Blue staining)
Lane M; AccuLadder™ Protein Size Marker (Low)
Lane 1,3,5,7 ; Negative control (No-DNA)
Lane 2; Bioneer's Kit
Lane 4; Company B's kit
Lane 6; Company C's kit
Lane 8; Company A's kit
Company B's kit positive control DNA expression test
Figure 5. Company B's kit positive control DNA expression test (SDS-PAGE and Coomassie Brilliant Blue staining)
Lane M; AccuLadder™ Protein size marker (Low)
Lane 1,3,5,7; Negative control (No-DNA)
Lane 2; Bioneer's Kit
Lane 4; Company B's kit
Lane 6; Company C's kit
Lane 8; Company A's kit