The cell-free protein expression system is also known as the in vitro translation system. The cell-free protein synthesis system uses the target mRNA or DNA as the template, adds the substrate and energy required for the protein synthesis to the enzyme system from the cell extract, and synthesize the target protein in vitro. The cell-free protein expression system simulates in vivo cells and reproduces the intracellular protein transcription and translation process. It needs the existence of various materials required for protein synthesis, including energy, transcription factors, and translation factors, etc.
The system is particularly suitable for the expression of transmembrane proteins and toxic proteins. Its feature includes short cycle and high-throughput.
Even though the system has more than 10 years of history, it still has some technical difficulties. Currently in the global base, cell-free protein expression is mainly provided through Rothe, Promega and other companies using kit expression, which has only very limited conditions and be very expensive. Our company is the first company to master the full set of core technology of E.coli cell-free expression system in domestic market, all core components are produced in house, and the reaction system contains more than 40 ingredients, which are easy to be adjusted and optimized. Since the establishment of this platform in 2015, 162 proteins have been successfully produced with yield of mg/ml, which contains 99 transmembrane proteins with 1-12 transmembrane domains and toxic proteins that are difficult to express in traditional E.coli expression systems. We have also produced high molecular weight proteins (130 kDa -140 kDa) that contain multiple transmembrane domains.
Compared with the traditional intracellular protein expression system, the cell-free system has the following significant advantages:
Risk-free: We do NOT charge if we cannot deliver the protein.
|Steps||Project||Process||Cusabio Features||Lead Time|
|1||Plasmid construction and preparation||Codon optimization, gene synthesis||Multi-vector optimization
In order to improve the efficiency of mRNA translation, thereby increasing protein yield, we provide protein expression service using N-terminal peptide optimization in addition to conventional N-terminal fusion protein. The N-terminal peptide contains 6-11 amino acids, it’s the shortest additional amino acid sequence that we have designed.
|15-20 business days|
|The PCR amplification products are ligated to the pET vectors by restriction enzyme digestion|
|Recombinant plasmids are prepared in large quantities|
|2||Small-scale expression and optimization||Multi-condition optimization;
Determine the optimal reaction condition
|Multi-condition expression scheme
In cell-free expression system, we can express several different proteins simultaneously on the multi-well plate under a variety of different conditions. Thus we offer multi-condition optimization service.
|7-10 business days
(Additional 3 business days for multi-condition purification)
|3||Target protein expression and purification||Prepare 1-10ml large-scale expression based on the small-scale results|
|The target protein is purified by exploring different chromatographic conditions including ion exchange chromatography, size exclusion chromatography and others by using AKTA, and then determine the optimal purification method.||Multi-condition purification scheme (optional)
For transmembrane proteins, we provide different detergent purification services to determine the optimum buffer for your transmembrane protein. This purification scheme is most suitable for transmembrane proteins with bioactivity.
|4||Additional services (optional)||Charge||Tag-removal service||Flexible additional services
Customers can flexibly choose from a variety of additional services to their specific needs, e.g. Endotoxin removal, Filter-sterilization, Tag removal, Lyophilization, etc. Some are complimentary, and some require additional charge.
|3 business days|
|Free||Endotoxin removal, Filter-sterilization, Lyophilization (Note: Lyophilization and filter-sterilization can not be met at the same time)||2 business days|
|5||Quality Control||Testing of purity, concentration, etc. QC report is provided.||Detailed COA report
Detailed product data sheet and COA are provided for each project.
|3-5 business days|
|Total lead time||25-35 business days|
The following three items are proteins with 5, 6 and 7 transmembrane domains separately. Since the in vivo expression system is difficult to express multiple transmembrane proteins, or the yield is very low, CUSABIO use cell-free expression system to produce these three proteins. To increase the yield, we explored a variety of expression conditions for the customer. Figure 1, 2, and 3 have shown the small scale expression of these three proteins under different conditions, and we selected the optimal condition for the large-scale expression.
The project was a 9 transmembrane protein. The difficulty of this project was not only the large number of transmembrane domains, but also the high molecular weight (141.7 kDa). After multiple-condition optimization, we successfully produced the protein with high yield, which can be observed on SDS-PAGE.
The protein in this project had a very low yield. Through optimization of different N-terminal peptides, the yield was improved dramatically, as shown in Figure 5.
HTR1B is a membrane protein of the GPCR family. It contains 7 transmembrane domains. We successfully expressed this protein and did the functional activity test, and the result has shown that the protein is bioactive.
The Binding Activity of HTR1B (7TM) with GSTK1Activity :
Characteristic expression systems
pET-23a(+)-JT vector, efficient expression in vitro
Carrys T7 strong promoter, does not contain lac operon, no negative repressive effect, can express protein efficiently;
Compared with conventional 6xHis tag, the N-terminal 10xHis tag has a stronger binding ability in IMAC. The thrombin site makes it easy to remove the tag.
The C-terminal MYC-tag can be used for WB detection, and it has stronger sensitivity compared with His-tag.
Amp resistance screening.
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