The kit is a product for in vitro protein synthesis based on Escherichia coli cell lysate. This kit utilizes active components in the cell lysate, including ribosomes, translation factors, and enzymes, supplemented with energy sources, nucleotides, amino acids, inorganic salts, etc., to reconstitute a transcription-translation system in vitro. It expresses target proteins using DNA or RNA as templates. Cell-free protein expression enables rapid, flexible and high-yield protein production independent of living cells, and offers numerous advantages over traditional recombinant expression systems:
1. Fast protein expression: Target protein can be produced within 1-2 hours, and maximum yield is achieved in 8-24 hours.
2. High protein yield: Up to more than 3000 μg/mL protein.
3. Simple and flexible reaction: Only DNA or RNA template needs to be added to the reaction system; reactions can be performed in 96-well plates or centrifuge tubes.
Note: This product can express conventional proteins and is especially suitable for expressing proteins containing disulfide bonds.
Application
This product is limited to scientific research use by professional personnel. It shall not be used for clinical diagnosis or treatment, nor for food or pharmaceutical applications.
Protocol
1. Gene Construction
Gene construct design is critical for efficient protein expression. The construction strategy shown below is recommended.The target gene can be cloned into the positive control plasmid supplied with this kit (plasmid map is available via the QR code on the outer package label). The kit is also compatible with pET-9a, pET-23a, and other pET-series plasmids containing the T7 promoter but without the lac operator.
Schematic diagram of the positive control plasmid DNA sequence is shown below:
Note: Plasmids containing the lac operator (such as pET-28a) may significantly reduce protein yield and are not recommended for direct use.
Figure 2 Schematic diagram of positive control plasmid DNA sequence
2. Template Preparation
The Cell-Free Protein Synthesis Kit supports the use of DNA or mRNA as templates for recombinant protein expression. DNA templates may include plasmids, PCR products, or RCA products amplified by phi29 polymerase.
(1) Plasmid DNA: Obtain a suitable plasmid via gene synthesis or subcloning, and purify using column-based plasmid extraction kits.
(2) PCR product: Design primers with the forward primer approximately 200 bp upstream of the T7 promoter and the reverse primer approximately 200 bp downstream of the stop codon (including the T7 terminator). Amplify the template; the resulting linear DNA fragment can be used directly in the cell-free reaction without purification. The 200 bp flanking sequences protect linear DNA from degradation by endogenous exonucleases.
(3) RCA product: Perform rolling circle amplification (RCA) using phi29 polymerase and random hexamer primers. The amplified DNA product can be used directly in the cell-free reaction.
(4) PCR and RCA can be combined with Golden Gate and Gibson Assembly to greatly accelerate and increase the throughput of DNA template preparation.
(5) DNA templates must be accurately quantified before use. High-quality plasmid extraction kits are recommended to avoid RNase A contamination. Plasmids received from gene synthesis companies must be column-purified; otherwise, they cannot be used directly in cell-free reactions.
3. Cell-Free Protein Expression
(1) Reconstitute lyophilized Cell-free system solution A Max and lyophilized Cell-free system solution B with 300 μL and 500 μL RNase‑free water, respectively(or reconstitute lyophilized Cell-free system solution A Max and B with 1.5 mL and 2.5 mL RNase‑free water, respectively).A vortex mixer or pipette mixing can be used to achieve complete homogeneity.
(2) Calculate the required volumes of Solution A and Solution B based on the total reaction volume (volume ratio 1:2). Add all reagents to the reaction vessel (e.g., 2 mL round‑bottom centrifuge tube) on ice and mix well.Gloves and a mask must be worn throughout the procedure. Use nuclease‑free pipette tips and reaction vessels to avoid nuclease contamination.The cell‑free reaction system can be prepared according to the table below:
Table 2 Reaction System Composition
| Component | Final Concentration | 50 μL System | 100 μL System | | Cell-free system Solution A Max | 30% | 15 μL | 30 μL | | Cell-free system Solution B | 60% | 30 μL | 60 μL | | Template DNA | 5–10 μg/mL | 5–10 μg/mL | 5–10 μg/mL | | Nuclease-free water | / | to 50 μL | to 100 μL |
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(3) Add template DNA to the reaction mixture. The recommended final concentration is 5–10 μg/mL; the amount can be further optimized.
(4) Incubate the reaction vessel in a shaking incubator or thermomixer for cell-free protein expression. The recommended reaction temperature is 25-30 °C.Lower temperatures reduce the protein synthesis rate but improve protein solubility. Maximum yield is typically achieved after approximately 8 hours; overnight incubation (16 h) is also applicable. Prolong the reaction time appropriately when using lower temperatures.
(5) Cell-free protein expression requires sufficient oxygen. When using a 2 mL round-bottom centrifuge tube, the reaction volume should not exceed 100 μL.The reaction can be scaled up proportionally using larger vessels (e.g., shake flasks) with a shaking speed of 200 rpm.
4. Detection
After completion of the reaction, take approximately 1 μL of the reaction mixture (for total protein) or supernatant (for soluble protein) and analyze target protein expression by SDS-PAGE.
5. Positive Control
The kit includes a sf-GFP (super-folder GFP) plasmid as a positive control, allowing direct visual observation of results.Upon successful expression of sf-GFP, the cell-free reaction mixture will show a distinct green color.For precise quantification of sf-GFP, detection can be performed using a microplate reader (Ex/Em=485 nm/528 nm).
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