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)


Sizeexclusion 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