The prokaryotic expression system has the advantages of high expression level, simple operation, short cycle, easy large-scale and high-density culture and low cost. For the full-length antibody and glycoprotein biological drug, the folding of expression product polypeptide chain, the disulfide bond, the presence or absence of glycosylation and the type of glycosylation often affect the properties of the synthesis, secretion, biological activity, in vivo stability, and immunogenicity of the expressed product. Compared with other eukaryotic expression systems, the expression of the target gene in mammalian cells is similar to that of the native protein in the type and manner of the glycosylation, and can be correctly assembled into the multi-subunit protein.
Risk-free: We do NOT charge if we cannot deliver the protein.
|Steps||Project||Process||Cusabio Features||Lead Time|
|1||Plasmid construction||Codon optimization; gene synthesis||Multiple vectors optimization, More options for customers
In order to improve the success rate of expression and achieve higher yield, in addition to conventional N-terminal fusion protein expression, we also provide C-terminal fusion protein, which retains the bioactivity of the protein while ensuring high purity.
|15-20 business days|
|The PCR amplification products are ligated to the vectors e.g. pSec series, pCMV series and pcDNA series vectors|
|Transform TOP10 E.coil competent cells|
|Obtain the correct recombinant plasmid|
|2||Small scale expression||Prepare the transfection grade recombinant plasmid in large quantities||Optimization of transfection conditions
Set different transfection conditions, select the optimal experimental conditions according to the test results.
|9-11 business days|
|Transient transfect HEK293, CHO and other cells|
|Detect expression products|
|3||Scale up expression and purification||Scale up the culture cells and transfect||Multi-condition expression scheme
According to the protein localization and the best experimental conditions in the small test expression, select different cell lines and different ways of transfection, which can increase the expression quantity, greatly improve the protein expression.
|8-9 business days|
|Explore different chromatographic conditions including ion exchange, hydrophobic and others by using AKTA, and then determine the optimal purification method.|
|4||Additional services (Optional)||Charge||Tag removal by restriction digestion||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||Filter-sterilization; Endotoxin removal; Lyophilization (Note: Lyophilization and Filter-sterilization can not be met simultaneously)||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||35-45 business days|
It is well known that the expression yield of mammalian cells is relatively low, the protein was optimally expressed with our mammalian expression system, the target band can be observed by SDS-PAGE analysis of the culture supernatants. The purified protein expression level can up to 10 mg/L. The theoretical molecular weight of the protein was 42 kDa, and the protein is modified with glycosylation by SDS-PAGE, which was confirmed by the examination of LC-MS/MS.
Three full-length antibodies were transfected into CHO cells using our vector, after SDS-PAGE detection, the bands were observed in the supernatant of the culture medium, the expressed level was up to 100 mg/L.
Characteristic expression systems
pSecTag2A-JT Vector+HEK293 Cell efficient expression
Carrys CMV strong promoter, containing IgK signal peptide which can enhance the secretory quantity of the protein
Compared with conventional 6xHis tag, the N-terminal 10xHis tag has a stronger binding ability in IMAC. Meanwhile the thrombin site makes it easy to remove the tag to obtain untagged protein.
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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