Cell free protein expression kit - disulfide bond enhanced version - freeze-dried powder

Item Number：P0243L

Storage conditions：-80℃，1yeareffective

Dry ice transportation. After opening, store the cell-free protein expression response components at -80 ℃. To avoid repeated freeze-thaw cycles, liquid A and liquid B can be separately divided according to the reaction amount, frozen with liquid nitrogen, and then stored at -80 ℃.

Product components

Product Description

The Cell Free Protein Synthesis Kit is aA product based on E. coli cell lysate for in vitro protein synthesis. This product utilizesActive substances such as ribosomes, translation factors, enzymes, etc. in cell lysate are supplemented with energyNucleotides, amino acids, inorganic salts, etc. are used to reconstruct transcription in vitro-Translation system. By DNA orRNA serves as a template to express proteins. Cell free protein expression is a type of expression that is independent of live bacteria,Being able to express proteins quickly and flexibly in large quantities, compared to traditional recombinant expression systems, it has many advantagesMultiple advantages:

1. Fast protein expression, the target protein can be expressed within 1-2 hours, and the maximum expression can be achieved within 8-24 hoursof large number.

2. Proteins with high expression levels, up to 3 mg/ml or higher.

3. The reaction is simple and flexible, requiring only the addition of DNA or RNA templates to the reaction systemuse96 well plate or centrifuge tube for reaction.

Note: This product can express conventional proteins and is more suitable for expressing proteins containing disulfide bonds.

Scope of application

The product is limited to scientific research by professionals and cannot be used for clinical diagnosis or treatment, nor for food or medicine.

Operating instructions

1. Gene construction

The construction of target genes is crucial for protein expression, and it is recommended to use the construction method shown in the following figure. The target gene can be constructed onto the positive plasmid provided with this kit (plasmid map can be found in the scanned QR code file on the outer packaging label), as follows:; It is also compatible with PET series plasmids such as pET9a and pET-23a that contain the T7 promoter but do not contain the lactose operon (lac).

Note: Plasmids containing the lac lactose operon (such as pET28A) can have a significant impact on yield and are not recommended for direct use.

The schematic diagram of the DNA sequence of the positive plasmid is shown below：

2. Template preparation

The cell-free protein synthesis kit can use DNA or mRNA as templates to express recombinant proteins. The DNA template used can be a plasmid, a PCR product, or a product of RCA rolling loop amplification using phi29.

2.1 PlasmidsSynthesize plasmids suitable for cell-free reactions through gene companies or subclones, and extract the plasmids using column purification;

2.2 PCR productsDesign primers, with the forward primer located about 200 bp upstream of the T7 promoter and the reverse primer located about 200 bp downstream of the stop codon (including the T7 terminator), to amplify the template. The obtained linear DNA fragment can be directly fed into a cell-free reaction system without purification. The function of the upstream and downstream 200 bp bases is to protect the linear DNA fragments from degradation by endogenous exonucleases.

2.3 RCA productsUsing phi29 polymerase and random six primers for rolling loop amplification of RCA, the obtained DNA product can be directly used in cell-free reaction systems.

2.4 PCR and RCA can be combined with Golden Gate and Gibson Assembly to greatly improve the speed and throughput of DNA template preparation.

2.5Accurate quantification is required before using DNA templates; It is recommended to use high-quality plasmid extraction kits for plasmid extraction to avoid introducing RNase A; The plasmid returned by the gene synthesis company needs to emphasize the use of column purification method, otherwise it cannot be directly used for cell-free reactions.

3. No cellular protein expression

3.1Calculate the required A and B solutions (volume ratio 1:2) based on the total amount of the reaction system, add the reagents to the reaction vessel on ice (e.g. 2 ml round bottomed centrifuge tube), and mix well. Gloves and masks should be worn throughout the operation process, and enzyme free pipette tips and reaction containers should be used to avoid introducing nucleases.

The cell-free reaction system can be mixed and prepared according to the following table:

3.2Add template DNA to the reaction system, with a recommended final concentration of 5-10 μ g/ml for DNA template addition, which can optimize the amount of DNA template added.

3.3Place the reaction vessel in a regular shaker or constant temperature mixer for cell-free protein expression, with a recommended reaction temperature of 25-30 ℃. Lowering the temperature will decrease the rate of protein synthesis, but will increase the solubility of the protein. Generally, the maximum protein yield can be achieved after about 8 hours of reaction, or overnight reaction for 16 hours. When the reaction temperature decreases, the reaction time should be appropriately increased.

3.4Cell free protein expression requires sufficient oxygen, and when using a 2 ml round bottomed centrifuge tube as the reaction vessel, the reaction system should not exceed 100 μ l. Cell free protein reactions can be proportionally amplified, and the amplified reaction system requires the use of larger reaction vessels, such as shake flasks, while ensuring the rotation speed of the shaker (200 rpm).

4. Testing

At the end of the reaction, take about 1 μ l of reaction solution (detect all proteins) or reaction supernatant (detect soluble proteins), and conduct SDS-PAGE protein gel electrophoresis to detect the expression of the target protein.

5. Positive control

This kit contains a green fluorescent protein sf GFP plasmid as a positive control, which can be directly observed by the naked eye for reaction results. After successful expression of sf GFP, the cell-free response system will show a clear green color.

To accurately quantify sf GFP, an enzyme-linked immunosorbent assay (ELISA) reader can be used for detection (Ex/Im=485/528 nm).