CubeServices Protein Purification
Cube Biotech Services for protein purification are open to any small to mid-scale, non-GMP protein purification project, with a focus on the demanding class of membrane proteins. Our service includes a thorough discussion and counselling of your project, so that the methods used provide optimal conditions for the purification of your protein of interest. Protein purification methods range from affinity chromatography using various tags including His, Strep, Rho1D4, and others, classical chromatography such as ion exchange and size exclusion for polishing to protein-specific affinity chromatography which takes protein purification to the next level and allows for the enrichment of active protein conformations. Protein purification can be booked as a separate service package. We can establish protein purification protocols or optimize existing methods. As a starting material, we accept most procaryotic or eucaryotic cell pellets, cell lysates, or cell culture supernatants. Alternatively, the full package of expression optimization and purification is available.
1. Starting materials
Depending on the expression host or expression vector used, proteins need to be purified from the cytoplasm, the (bacterial) periplasm, cell membranes, or cell culture supernatants. We have established optimized protocols for each of these applications, which we apply to the particular host-vector combination.
2. Buffer and detergent screening
For soluble and membrane proteins, the optimal buffer conditions for stability are determined. In the case of membrane or membrane-attached proteins, detergents are required to extract the proteins from bacterial or eukaryotic membranes. Detergents are optimized for protein stability during extraction and purification steps. If proteins are expressed as inclusion bodies, resolubilization and refolding is done, including a screen for the optimal detergent and buffer conditions.
3. Affinity purification
Purification of proteins fused to tags like His, GST, or Strep tags is performed using our high-quality affinity purification matrices. For membrane proteins, we recommend to use the Rho1D4 system. Purification using other tags (e.g. FLAG) is also available upon request. Protein-specific matrices can be synthesized by coupling specific ligands to affinity matrices, enabling a separation of the active protein fraction. Purification conditions are optimized in small scale, and upscaling can be done up to mg amounts, depending on the protein of interest. Membrane proteins can optionally be integrated into nanodiscs to increase stability. Proteins are concentrated and aliquoted according to customer requirements. A thorough documentation of all steps is provided, including SDS-PAGE and Western Blot data.
4. Further purification steps for crystallization-ready proteins
To obtain highly pure, homogeneous protein fractions suitable for e.g. protein crystallization experiments, a second or third purification step can be performed, using, for example, ion exchange chromatography or gel filtration. Purification conditions are optimized in small scale, and upscaling can be done up to mg amounts depending on the protein of interest. Proteins are concentrated and aliquoted according to customer requirements. A thorough documentation of all steps is provided, including SDS-PAGE and Western Blot data.
5. Protein characterization
Purified proteins can be tested ligand binding and structural integrity, or even enzymatic activity. Learn more