Protein production

Steps 1 and 2: construct design and molecular biology 

Our experienced protein scientists collaborate with you to design constructs aimed at achieving the highest success for your project. Leveraging a combination of our extensive expertise, current literature, limited proteolysis analysis and advanced bioinformatic tools like AlphaFold2, we optimise sequences – for mutants, fusion and truncated proteins and more – for various expression hosts. Our customised molecular biology services include efficient parallel cloning in a versatile in-house vector panel, offering flexibility in tag selection and removal.  

Step 3: protein expression 

Nuvisan´s extensive experience with various host systems, including E. coli (e.g., BL21(DE3), Arctic), mammalian cells (e.g., CHO, HEK293, Expi293F) and insect cells (e.g., Tni, Sf9), helps ensure optimal expression of our generated vectors. Our multi-parallel optimisation approach identifies ideal production conditions for up-scaled expressions. We offer capabilities ranging from small-scale (plate format), mid-scale, to large-scale expression, tailored to the optimal conditions and requirements of your target protein. 

Large scale expressions can be carried out in up to 100 L fermenters (E. coli) or 4-16 L wave bioreactors for insect cells, which can result in up to grams of recombinant proteins to support e.g. large screening campaigns. With our smaller 1-L and 2-L fermenters, we can run process optimisations to find the best expression conditions for bioreactor approaches (batch, fed-batch processes) for your project.

Step 4: protein purification 

Our protein scientists optimise purification processes using multi-parallel expressions in a 96-well format and various purification matrices (e.g., IMAC and glutathione sepharose). Our streamlined approach includes target-specific buffers at the small-scale stage, with optional use at the mid-scale stage, enabling early estimation of protein yields and easy adaptation to large-scale production. Equipped with over 10 state-of-the-art ÄKTAT instruments, we efficiently run multiple purifications in parallel, employing diverse chromatography techniques, from affinity and ion exchange to hydrophobic interaction and gel filtration. This combination ensures high-throughput, high-quality protein purification. 

Step 5: quality control  

At every step of our production pipeline, we implement quality control measures to monitor and optimise the protein production and purification process. All purified proteins undergo an in-depth quality control regimen based on SDS-PAGE and MS, with additional biophysical and biochemical assays available. Our quality control methods include: 

• SDS-PAGE and MS to assess purity and integrity 
• DLS, SEC-MALS and F-SEC to analyse oligomerisation and aggregation states 
• TSA, nanoDSF, NMR and CD to evaluate protein folding and stability, offering the most stable construct for crystallisation trials 
• SPR, ITC, NMR and MST to examine binding activity 
• Biochemical, NMR-based or MS-based assays to measure enzymatic activity 
• Endotoxin testing.