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Research & Development
Recombinant Proteins
Primm is engaged in several industrial collaborations related to the development and exploitation of expression systems and production processes based on recombinant DNA technology. This service includes cloning of DNA deriving from any biological source (prokaryotic or eukaryotic) and type (genomic, cDNA, PCR products, or plasmid fragment) into a standard vector, DNA sequencing of the gene, and eventual development of recombinant strains (Bacterial, yeast, insect or mammalian cells) expressing heterologous proteins. The service also includes preparation of the strain, protocols for fermentation, downstream processing, purification, and characterization of the biological activity of the proteins produced. Primm also collaborates on pre-clinical and clinical development of specific biopharmaceuticals.
ORGANISM EXPLOITED
Microbial cells
Escherichia coli is the most popular system to produce recombinant proteins. We use several expression strains and vectors representing the state of the art of these technologies and our de novo developed systems. Primm also exploits production systems based on recombinant yeast cells such S. cerevisiae and P. pastoris. This production systems may offer a useful alternative to bacteria-based systems.
Mammalian cells
Mammalian cell lines offer several advantages for expression of heterologous proteins. Since transcription, translation, and post translational modification processes are conserved among higher eukaryotes, eukaryotic proteins produced in mammalian cell may be functional and well suited for a variety of structure-function assays. These services include transfection of vector DNA into mammalian cells for stable or transient expression of gene products. A variety of transfection methods and assay systems are available.
PRODUCTION STEPS
Host and vector selection
- Studies for the best choice of the couple host/vector
- In silico cloning
- Cloning in the appropriates vectors of the gene of interest
- Transformation/trasfection of the chose host cells
- Screening of the positive clones
Protein Expression Optimisation
- Expression system optimisation to overcome low protein production and/or insoluble product
- Parameters explored: induction agents, time course evaluation of protein production, time course evaluation of soluble vs. insoluble product, induction at various cell densities, regulation of temperature and evaluation of post induction harvest time
- Protein refolding from inclusion bodies
Protein purification
- Development of de-novo methods for natural-source and recombinant proteins
- Improvement of existing protocols with alternative paradigms / chemistries and the identification of cost-saving methods
- State-of-the-art protein purification systems and available chemistries
PROTEIN ANALYSIS
Protein identification
- MALDI-TOF and ESI mass spectrometry
- N-terminal sequencing · Peptide mapping
- Isoelectric focusing and western blot (WB)
- Bioassays
Protein characterization
- Purity determination by HPLC, SDS-PAGE, WB
- Determination of the host proteins and nucleic acids contaminants
- Pirogens content
FACILITIES
- 2 to 15 L fermenters
- High volume ultrafiltration systems
- FPLC and HPLC systems
- Protein characterization systems
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