Production of recombinant proteins
Flexible and tailored approach to meet client’s needs
- Customized design of protein production strategy
- Production of recombinant proteins according to protocols established in-house
- Production of recombinant proteins according to client-supplied or published method
Project divided into phases
- Feasibility studies for production of particular protein
- Broad matrix of expression conditions for initial screening – further customization available
- Pilot overexpression and purification
- Optimization of established production protocols
- Small to large-scale production (from microgams to grams of target protein)
- Batch-to-batch reproducibility
- Each phase of the project executed separately or in paralel if needed
- Decision GO / NO GO for further stages of the project at completion of each phase
- Flexibility in the execution plan (e.g. additional phases)
- FTE and FFS collaboration models
Deliverables
- Required quantity of recombinant protein of highest possible purity (˃ 90, 95, 98% ), high homogeneity, desired oligomeric state, and proper fold
- Tagged with selected tag or untagged
- Proteins in solution or in lyophilized form
- High concentration available (10-50 mg/mL)
- Cell paste containing overexpressed proteins for client’s downstream applications
Extensive expertise of our scientific team, including hands-on experience with a broad range of target proteins, provides a strong basis for meeting client’s expectations
Broad range of targets for various therapeutics areas and downstream applications:
- Oncology
- Inflammation
- Vaccines development
- Neurodegenerative therapies
- Fibrosis
- Enzymatic assays
Variety of proteins
- Different molecular functions: kinases, ATPases, DNA demethylases, transcriptional regulators, etc.
- Diversity of biological role: angiogenesis, regulation of cell cycle, response to DNA damage
- Viral, bacterial, fungal, animal and plant proteins
- Virus-like particles (VLPs)
- Subunits of bacterial toxins
- Single domain antibodies (nanobodies, VHH)
- Protein complexes and membrane proteins
- Intrinsically unstructured proteins
Customized production
- From tens of µg to hundreds of mg of high quality, purified protein
- Purification from soluble fraction or refolded proteins
- Protein complexes – in vivo co-expression of target subunits or in vitro complexation
- Nucleic acid-binding proteins with low nucleic acid content
- Non-radioactive isotope-labeled proteins, and other modifications (e.g. biotinylation, phosphorylation).
- Low endotoxin and sterile proteins
Various expression systems for your choice
E. coli
Relatively low cost of production
Limited folding and post-translational modifications
- A range of bacterial strains
- Cytoplasmic and periplasmic overexpressionCulture flasks with capacity up to 100L / week
- Scale of production 0.5 – 200 mg (gram scale available)
- Timeline: 4 – 10 weeks
Insect cells (BEVS)
Proper folding, post-translational modifications
Suitable for expression of large proteins >100 kDa, capable of producing cytotoxic proteins
- Various insect cell lines
- Different baculovirus generation systems
- Transient expression
- Intra and extracellular overexpression
- Culture flasks with capacity up to 40L / week
- Scale of production 0.5 – 200 mg (gram scale available)
- Timeline: 6 – 12 weeks
Mammalian cells
Proper protein folding
Desired post-translational
modifications
- Different mammalian cell lines
- Transient expression / Infection with baculovirus
- Intra and extracellular overexpression
- Culture flasks with capacity up to 6 L / week
- Scale of production 0.5 – 200 mg
- Timeline: 5 – 10 weeks
Diverse purification strategies
Isolation and purification of proteins from separated cellular fractions
- Soluble or insoluble
- Cytoplasmic, periplasmic, membrane-bound or secreted
Various cell lysis methods
- Freeze-thaw
- Sonication
- Homogenization
- Detergent solubilization
- Hypotonic solutions
Affinity chromatography based on
a wide range of different tags
- His-tag (cross-linked agarose chelating metal ions; Ni2+, Zn2+, Co2+)
- GST-, MBP-, Strep-, Twin-Strep, FLAG-, c-Myc tags
- Protein A chromatography
Ion exchange chromatography
- Various types of prepacked columns (weak/strong ion exchangers)
Size–exclusion chromatography
- Selection of the SEC columns with various resolution in specific MW ranges (1 kDa – 5 MDa)
Additional methods
- Heparin chromatography
- Hydrophobic interaction chromatography (HIC)
- Concanavalin A (ConA) affinity chromatography
- Streptavidin Mutein Matrix
Customized in-house prepared columns
Non-chromatographic methods
- Ammonium sulfate precipitation
- Heat treatment for thermally stable proteins
- Centrifugation in sucrose gradient
Various strategy of protein refolding
- Dialysis
- Rapid dilution
- Pulse – refolding
- On-column refolding
Comprehensive and detailed analyses of protein quality
Standard Quality Control
- Identity – Peptide mapping (MS) and / or N – terminal sequencing (Edman degradation), Western Blot analysis
- Purity – SDS-PAGE followed by CBB or silver staining, optionally RP-HPLC
- Nucleic acids contamination – Spectrophotometric methods
- Concentration – Absorbance at 280 nm, Bradford, BCA, densitometric methods
- Homogeneity and oligomeric status – HPLC-SEC with a selection of analytical columns
- Overall fold – nanoDSF or DSF analyses
- Verification of Batch-to-batch consistency – SDS-PAGE, HPLC-SEC, nanoDSF / DSF, MS analyses
Optional
- Determination of endotoxin level
- Determination of isotope enrichment level
- Determination of biotinylation site and level
- Analysis of protein complexes
- Sterility testing
- Determination of protein’s activity
- Intact mass, PTMs
- Long-term storage stability studies
- Binding interactions with small molecules
Extensive troubleshooting – fast assessment of expression and purification feasibility and potential for further improvement
Optimization of the expression conditions to overcome inefficient production
- Flexibility of the construct design (codon optimization, tags, mutations, deletions, construct boundaries)
- Extensive expression tests (different bacterial strains and eukaryotic cell lines available, screening of a wide range of parameters such as media, temperature, induction conditions, transfection reagents, MOIs, time of protein production)
- Expression with solubility tags/chaperons/solubility enhancers
Development and optimization of purification protocols
- Comprehensive search for available and the most recent literature
- Top of current protein purifications technologies
- Screening for the optimal purification buffer
- Protocols to prevent or limit aggregation
- Strategies to reduce degradation of target protein
- Various endotoxin removal methods
Why Selvita?
Necessary prerequisites to successfully execute the requested projects
- Advanced equipment used during a project
- Team of highly skilled scientists (40% of the team with PhD degree)
- Know-how and extensive expertise in the production of various classes of proteins
- Each project supervised by experienced Team Leader (>10 years of experience)
Communication
- Regular updates (communication of the progress either by email or during the
T-con) - Comprehensive final reports
- Confidentiality
- Integrated assistance from idea to purified protein
- Technical support after completion of the project
Others
- Developed and well established protocols for the production of over 200 recombinant proteins
- More than 150 projects per year
- Full transparency of the whole process
- High rate of successful projects (˃90%) delivered within the agreed timeline
- High rate of returning Clients (>90%)
- Long-term collaborations with returning Clients
- Openness to collaboration with new Clients
- Collaboration both with academic as well as industrial scientific entities