Institute of Medical Biology PAN

We invite interested parties to contact us for an individual pricing assessment of services related to the integrated identification, screening, and production (up to a large laboratory scale) of recombinant proteins. We can provide professional assistance at both the advisory and execution levels.

A sample research and development pipeline, which can be fully carried out at our facility:

• Identification of genes encoding proteins with desired activities, found in nature, from genetic sequence databases
• Small-scale production (up to 100 µg) via in vitro translation
• Enzymatic activity testing using methods such as ELISA, hydrolysis of fluorogenic substrates, or bacterial growth inhibition in liquid cultures
• Selection of a favorable natural gene variant followed by in silico mutagenesis to enhance enzymatic activity, stability, affinity, etc.
• In vitro translation of mutated protein variants
• Re-evaluation of activity or thermal/chemical stability using the nanoDSF method or protein-ligand affinity (association and dissociation dynamics) using the BLI method
• Selection of the most advantageous mutated variant, vector construction, and cloning into an expression system
• Small-scale laboratory expression (up to 2 L of culture), optimization of homogenization/sonication conditions, and purification of recombinant protein (FPLC)
• Large-scale laboratory expression, bioreactor cultivation with a working volume of 3 to 16 liters. Optimization of cultivation conditions and preparation of the process for scaling up to the pilot scale.

Possible Areas of Application:

• Discovery of new proteins for industrial, medical, or research applications
• Verification of enzymatic protein candidates identified through bioinformatics analyses
• Adaptation of an enzyme with confirmed laboratory activity for industrial production
• Acceleration of research aimed at obtaining desired traits through targeted mutagenesis
• A technology park offering world-class scientific equipment for measuring properties, interactions, and effects of recombinant proteins (microscale thermophoresis, capillary electrophoresis, BLI, nanoDSF, qPCR, ultracentrifuge)

List of Selected Individual Research Services:

Service costs will be subject to individual pricing depending on the scope of services and the specific needs of the client. We are open to collaborative implementation of research and development projects, either as a consortium partner or as a research service provider.

Bioinformatics Services

• Searching for gene variants potentially encoding the desired protein in nucleotide sequence databases derived from natural sources
• In silico mutagenesis – targeted gene sequence modification based on known structures, related gene sequences, molecular docking, etc., aimed at obtaining a mutated protein variant with potentially improved functional properties

In Vitro Translation

• Production of up to 100 µg of protein directly in a test tube, bypassing a living expression host, using the ExiProgen™ (Bioneer) system. This method enables time- and cost-efficient screening of different enzyme variants on a microscale. It is limited to proteins that can be expressed in E. coli-based systems.
• Protein profile analysis of a sample on a microscale using capillary electrophoresis

Enzymatic Activity Assays

• Measurement of fluorogenic substrate hydrolysis
• ELISA tests
• Bacterial growth inhibition assays in liquid cultures
• Evaluation of protein impact on gene expression in bacterial cells or eukaryotic cell cultures in vitro using real-time RT-PCR

Interaction Measurements via BLI (Biolayer Interferometry) or Microscale Thermophoresis

• Affinity measurement: association and dissociation constants between proteins and ligands (protein or small-molecule)
• Antibody specificity and affinity testing
• Binding analysis between proteins and viral, virus-like particles (VLP), or nanoparticles
• Protein-nucleic acid interaction studies

Protein Stability Measurements Using nanoDSF (Nano Differential Scanning Fluorimetry)

• Determination of protein thermal stability
• Evaluation of resistance to selected chemical denaturing agents

Expression in Bacterial Systems

• Construction of expression vectors and transformation of expression host cells
• Shaken culture in volumes of 0.2 – 5 liters
• Bioreactor cultivation at 3 – 16 liter scale
• Optimization experiments in bioreactor cultivation and preparation for process scaling
• E. coli is the default expression system, but for larger projects, alternative expression systems may be considered

Protein Recovery and Purification from Biomass

• Sonication or mechanical homogenization of biomass to release intracellular recombinant proteins
• Selection of bacterial protease inhibitor cocktails for larger-scale production
• Renaturation trials for proteins trapped in inclusion bodies (if present)
• Purification of selected protein from lysate using FPLC, based on histidine tags or tag-free methods (ion-exchange, hydrophobic, or size-exclusion chromatography)
• Protein profile and purity analysis using SDS-PAGE electrophoresis, and specificity confirmation via Western blot (if specific antibodies are available or the protein carries a tag)
• Selection and optimization of buffer systems for storage and enzymatic reactions

Contact
Prof. Jarosław Dziadek, PhD, DSc
Phone: +48 42 27 23 633
E-mail: jdziadek@cbm.pan.pl