CubeServices Protein Expression and Purification
Protein Expression - From Gene to Protein
Cube Biotech Services for protein expression are open to any small to medium-sized non-GMP projects, with particular expertise in the demanding class of membrane proteins. Available expression hosts are E. coli and insect baculovirus systems (Sf9 / Trichoplusia ni). Alternatively, proteins can be expressed in E.coli cell-free lysates. Our service offering includes a thorough discussion and counselling of your project, making sure that the methods used provide optimal conditions for the expression and purification of your protein of interest. Any service package can be booked on its own, or in conjunction with others. Learn more about our protein services.
Expression constructs are tailored to the expression system of choice. We typically start with sequence-optimized, full-length genes expressing the wild type protein. In accordance to the requirements of the project, affinity tags and protease sites can be included
2. Expression screening and optimization
Each expression construct is optimized for protein yield with regard to
- Expression host (bacterial, insect, cell-free)
- Expression conditions (media, temperature, induction conditions)
- Promoter regions (strong/weak expression, tightness of control
- Type and position of affinity purification tag
- Optional: best-expressing domains, including a boundary screen of domain borders
Clear and thorough documentation is provided, including SDS-PAGE and Western Blot data.
3. Purification screening
Protein expression is first tested in small scale, high-throughput experiments to identify the optimal combination of
- affinity matrix or protein-specific matrix
- buffer conditions and pH
- eluent conditions and additives
- For membrane proteins: Detergent type and concentration
Small scale affinity purification followed by analytical gel filtration are performed to confirm tag accessibility, lack of unspecific interaction with the affinity matrix, correct folding and yield, and homogeneity of the resulting recombinant protein. A clear and comprehensive documentation is provided, including SDS-PAGE and Western Blot data.
4. Expression and purification scale-up
Using conditions optimized in step 3, the protein of interest can be purified to larger amounts, using affinity chromatography, ion exchange chromatography, and gel filtration methods. Customized affinity matrices can be synthesized on demand to separate the active, ligand-binding protein fraction. The purified protein is provided in aliquots as defined by the customer.
5. Protein characterization
Purified proteins can be tested ligand binding and structural integrity, or even enzymatic activity. Learn more