CubeServices Cell-free Protein Expression
Directly from DNA to protein - without any cell
Cube Biotech Services for cell-free protein expression are specifically suited for small- to medium scale expression projects, as well as high-throughput expression screening experiments. In combination with the nanodisc technology, even membrane proteins can be expressed in sufficient amounts for SPR and interaction studies. We use E.coli cell-free lysates which provide the highest yields and the most cost-effective solution. However, if other cell-free systems are preferred, we are open for discussions. All cell-free expression projects are non-GMP development projects.
- Expression construct screening
- Screening of expression conditions
- Reconstitution of membrane proteins into nanodiscs
- Small scale expression of proteins for interaction studies, or SPR
- Labelling of proteins with biotin, fluorescent labels, or isotope-marked amino acids for NMR
Cell-free expression can be combined with a range of other services, including protein purification and SPR measurements. 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.
1. Expression construct design and generation
Expression constructs are tailored to the expression system of choice. We typically start with sequence-optimized, full-length genes expressing the wild type protein.
2. Construct and expression optimization
In a small-scale screening experiment (50-200 µL), expression conditions are optimized with regard to
- Choice of expression vector / promotor
- Type and position of affinity purification tag
- Optimization of expression conditions to improve yields
- Optional: screening for best-expressing domain(s)
- Optional: screening of reaction conditions for disulfide-forming proteins
Additional options are available for membrane protein expression:
- No additives: Expression as insoluble pellets with subsequent refolding
- Using detergent: type and concentration of detergent
- Using nanodiscs: diameter of nanodisc, and composition of phospholipids
Clear and thorough documentation is provided, including SDS-PAGE and Western Blot data.
3. Purification screening
Protein obtained in the small scale experiments is purified via affinity magnetic beads, optimizing buffer and additive conditions. Customized affinity matrices can be used to separate the active, ligand-binding protein fraction. Clear and comprehensive documentation of this step is provided, including SDS-PAGE and Western Blot data.
4. Upscale of expression
Expression yields are very much protein dependent. From the results obtained in step 2 and 3, actual expression yields for the protein of interest can be calculated, and upscaling can be done to obtain the required protein amounts.
5. Purification Upscale
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.
6. Protein characterization
Purified proteins can be tested ligand binding and structural integrity, or even enzymatic activity. Learn more