BioActive™ CD Molecule Protein Production

Seattle Genova offers our customers excellent protein production services that keep the bioactivities of your proteins just as they were in nature. To achieve this, we can offer both nature protein purification services and recombinant protein production services. We have developed very specialized protocols to make sure the produced proteins will maintain their biological activities. And our well-developed bioassays can ensure comprehensive activity validations before we release the proteins.

The cluster of differentiation (or cluster of designation), always abbreviated as CD, is a protocol utilized for the identification and investigation of cell surface molecules existing in white blood cells (cells of the immune system). The cluster of differentiation nomenclature was proposed for the category of the many monoclonal antibodies (mAbs) produced by different laboratories around the world against epitopes on the surface molecules of leukocytes. Since then, the aim of clusters of differentiation has widened to various other cell types, and for humans, the CD is numbered up to 350 most newly (as of 2009).

 The cluster of differentiation nomenclature was proposed and organized in the 1st International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA), which was carried in Paris in 1982. Immunologists produced vast umbers of monoclonal antibodies reactive with leukocyte cell-surface molecules, each with various associated nomenclatures. In the lack of comparative studies, it was often difficult to tell if the same molecule was recognized by more than one antibody. The strategy of the workshops was to code antibodies and send them to multiple participating laboratories for blind analysis against multiple cell kinds. The data were collated and assessed by the statistical procedure of "cluster analysis." This analytical technique identified clusters of antibodies with very related patterns of binding to leukocytes at several phases of differentiation: hence the "cluster of differentiation" (CD) nomenclature. The cluster of differentiation nomenclature enabled the scientific community to communicate outcomes in a universal language.

Working process 

1.Design sequences

We do sequence analysis and codon optimization for the expression system that will be used. The designed sequences are then synthesized to generate a DNA template.

2.Cloning and Expression

The target gene is cloned into expression vectors for the mammalian, insect or E.coli cells. Multiple N-terminal signal peptides for protein secretion and N-terminal or C-terminal tags for protein affinity purification are available to choose from. The recombinant construct is confirmed by DNA sequencing.

The plasmid cDNA is extracted from the cells and transfected into 100 ml of E. coli or 50 ml of mammalian or insect suspension cells. The target protein in the cell lysate and supernatant is analyzed via SDS-PAGE and/or western blotting using an anti-tag antibody.

3. Bioactivity Testing

We can develop bioassays to validate the activity of the proteins we prepared. Only those proved to be active are qualified for release. Otherwise, we will start over to try different protocols until the bioactive protein is prepared.

4.Large-scale gene expression and protein purification

The expression vectors are prepared with endotoxin-free plasmid extract kits and are transfected into 2 liters of E. coli or 500 ml of mammalian or insect suspension cells. 

After the cell culture period, the target protein is purified via affinity chromatography. 

Concentration determined by: Bicinchoninic acid assay (BCA)

Purity determined by : SDS-PAGE with Coomassie blue staining

 The protein is desalted into 0.2 µm filtered 1x phosphate-buffered saline (PBS), pH 7.4. The proteinn can then be aliquoted, lyophilized, and labeled upon request.

5.Bulk production (optional)

Large-scale gene expression and purification based on the customer's desired amount of purified protein.

Service Highlights 

√High Purity

√Biological Activity

√Lot-to-lot Consistency

√Low Endotoxin

√Long-term storage feasible

Deliverables

SDS-PAGE, WB and the bioactivity validation data are sent to our customers. The purified proteins are also shipped to our customers for them to test internally.

References

1.CHAN, J. K. C.; NG, C. S.; HUI, P. K. (1988). "A simple guide to the terminology and application of leucocyte monoclonal antibodies". Histopathology. 12 (5): 461–480.

2.Zola H, Swart B, Banham A, Barry S, Beare A, Bensussan A, Boumsell L, D Buckley C, Bühring HJ, Clark G, Engel P, Fox D, Jin BQ, Macardle PJ, Malavasi F, Mason D, Stockinger H, Yang X (2007). "CD molecules 2006--human cell differentiation molecules". J Immunol Methods. 319 (1–2): 1–5.