Heterologous Protein and Peptide Production Core

Coordinators:

Prof. Elio Pizzo (elipizzo@unina.it)

Prof.  Eugenio Notomista (notomist@unina.it)

Prof. Valeria Cafaro (vcafaro@unina.it)

Prof. Andrea Bosso (andrea.bosso@unina.it)

Prof.  Rachele Isticato (isticato@unina.it)

The Heterologous Proteins and Peptides Production Core of the Department of Biology of the University of Naples Federico II provides consultancy, assistance and service for the production and purification of recombinant proteins in different host organisms such as Escherichia coli, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae and CHO cells.

The Heterologous Protein and Peptide Production Core holds the following areas:

Our technology allows us to express a wide range of proteins including enzymes, growth factors, antibodies, structural proteins and bioactive peptides.

1. Production of recombinant proteins: We design the gene(s) coding sequence for the proteins of interest within the appropriate DNA vectors. Our platform guarantees high efficiency of expression and protein yield.
2. Protein purification: We set the purification strategy in advance and apply well-established purification methods, including affinity chromatography, ion exchange chromatography, gel filtration or reverse phase using our automated HPLC and FPLC systems to obtain highly pure and functional products.
3. Process optimization: We offer optimization services to improve protein expression, stability and solubility, adapting our processes to the specific needs of each project.
4. Analysis and characterization: We provide detailed analyzes of the proteins produced, including tests for purity, enzymatic activity, secondary structure and thermal stability.
5. Labelling of peptides and proteins: We develop strategies to label proteins and peptides at specific sites with commercial labels or fluorophores and with environment sensitive fluorophores useful for binding and kinetic studies, conformational analysis, membrane interaction, cell internalization etc.
6. Chemical modification of peptides and proteins: We offer optimization services to improve solubility and/or stability by chemical modifications (e.g. pegylation, cyclization, etc.).
7. Immobilization of peptides and proteins: We develop strategies to optimize protein and peptide immobilization onto surfaces also by the addition of spacer peptide sequences (random coil peptides).
8. Display of heterologous proteins on bacterial spores: We use the “spore surface display technology” for the presentation of proteins of interest, enhancing their stability and functionality.

Equipment:

• Several engineered Escherichia coli strains such as:

1. 1. BL21 (DE3): A very popular strain for the expression of recombinant proteins. This strain contains the DE3 lysogen, which brings the T7 RNA polymerase gene under the control of the lacUV5 promoter.
   2. BL21 (DE3) pLysS e BL21 (DE3) pLysE: Variants of strain BL21 (DE3) that express T7 lysozyme to reduce the toxicity of recombinant proteins.
   3. Rosetta (DE3): A strain that expresses rare tRNAs to enhance the expression of recombinant proteins derived from eukaryotic organisms.
   4. C41 (DE3) e C43 (DE3): Strains derived from BL21 (DE3) selected for their ability to express toxic proteins.
   5. Origami (DE3): A strain engineered to facilitate the formation of disulfide bonds in recombinant proteins.
   6. RIL (Rare tRNA Improved Levels): they are strains designed to enhance the expression of heterologous proteins.

• Eukaryotic cells lines to preserve any post-translational modifications in the recombinant protein;
• HPLC and FPLC chromatographic systems;
• Plate readers for sample characterization;
• Freeze dryer for sample preparation;
• Thermostatic shakers and incubators.

Additional Services

• Endotoxin test (LAL assay)
• Protein characterization (Western Blot, HPLC, mass spectrometry, cytotoxicity)
• Tag removal (Protease digestion)

ACKNOWLEDGEMENTS

All the users and collaborators of the Heterologous Proteins and Peptides Production Core are obligated to acknowledge the core in publications: “The authors acknowledge the Heterologous Proteins and Peptides Production Core at Department of Biology of University of Naples Federico II.