IBBA – IBISBA-IT

Consiglio Nazionale delle Ricerche

Istituto di Biologia e Biotecnologia Agraria

Optimization of protein expression and secretion in eukaryotic hosts. Design for the expression of toxic polypeptides. Environmental metagenomics for identification of novel microorganisms

Area of Research

Biogenesis and evolution of eukaryotic cellular compartments, with particular interest in the synthesis and accumulation of seed storage proteins (biogenesis of protein bodies in cereal seeds; nutritional qualities; allergenic/anti-nutritional).
Mechanism of Quality Control in the Endoplasmic Reticulum (ERQC) and ER associated degradation (ERAD).
Modulation of ER quality control (ERQC). Molecules modulating ERQC and/or ERAD have potential as: i) broad spectrum rescuers of the secretion of responsive mutants of glycoproteins that cause rare diseases; ii) broad spectrum antivirals; iii) anticancer chemotherapics. Structural (electron cryomicroscopy and X-ray crystallography) and functional analysis of ERQC/ERAD glycoproteins and their complexes with substrate glycoproteins. Biological and biochemical hypotheses are generated, then tested with in vitro and in cellula assays, and in vivo experiments using the plant as a model system.
Unfolded Protein Response (UPR) during seed development and storage protein accumulation in normal and stressed conditions.
Biogenesis of membrane proteins (channels/transporters; tail-anchored proteins) studied with a cross-kingdom and Comparative Cell Biology approach, mainly focused on tonoplast versus lysosomal membrane residents.
Planning of strategies to optimize the production of recombinant proteins and peptides of pharmaceutical/food/industrial interest in plant systems used as bioreactors. Exploiting the ER chaperones to sustain recombinant protein synthesis.
Genomics, transcriptomics, and metagenomics in livestock and animal products. The activities span from the study of genes and mechanisms that control the livestock production and health to the study of microbiota in livestock and animal products, with special focuson dairy products.
Environmental and agricultural metagenomics, with particular interest in water, soil, plant and rhizosphere communities characterization.
Molecular Microbiology for food quality and safety and animal health, especially developing methods for pathogens detection and characterisation

Expertise and Main Technologies

CELL BIOLOGY EDUCATED PROTEIN DESIGN AND TESTING
DESIGN strategies to produce high levels of recombinant proteins and peptides in plants as bioreactors according to an in silico pipeline
The known structural features of the protein of interest (POI) are used for in silico modelling of the POI, and the choices of vector, target sequence, codon-usage, expression system and purification strategy are tailored to the required POI properties (e.g. stability, purity, or alleviation of allergenic/toxic features).
TESTING the different strategies by transient expression in plant systems
In vivo test of the engineered POI using rapid, reliable transient expression systems (plant cell protoplasts, leaf agroinfiltration, yeast or bacterial expression), depending on the POI features. POI stability (half-life) and subcellular localization as well as co- and post-translational modifications are monitored by microscopy and biochemical/biophysical analysis.
Optimisation of recombinant POI(s) sequence(s), expression system and expression strategy is carried out by testing for POI yield, its intracellular localization/stability and monitoring host physiology and/or transcriptomics of the expression host during POI expression (for plants, this is performed in different tissues).
The best protein engineering strategy will be used for stable production of POI in plants.
Vectors for transient and permanent expression containing the POI of interest are provided to the customers as the final product.
For details see also https://www.youtube.com/watch?v=Lj3v6G6YTY4
FUNCTIONAL CHARACTERISATION OF PROTEINS
In silico modelling of recombinant proteins for optimization of their production in plants, including post-translational modifications and prediction of subcellular localisation
Analysis of:
- Folding: resistance to proteolytic digestion, disulfide bond pattern, solubility, binding to conformation-dependent antibodies
- Oligomerization: velocity gradient centrifugation in different biochemical conditions; dynamic light scattering (DLS); SEC-MALS
- Glycosylation: analysis with glycan-specific antibodies and deglycosylating enzymes; mass-spectrometry analysis (glycomics)
- Targeting and sorting: density gradient subcellular fractionation; fluorescence microscopy
PROTEIN/NUCLEIC ACIDS STRUCTURAL BIOLOGY SERVICE
Structure determination using experimental and computational resources (virtual machine), based on X-ray diffraction and/or Cryo-electron microscopy.
METAGENOMICS AND METABARCODING
Based on the following:
- Extraction of genomic DNAs directly from the environmental samples;
- Preparation of metagenomic libraries;
- Shotgun metagenomic sequencing by Next generation sequencing using Illumina technology (Metagenomics);
- Bioinformatics analyses (meta-assembly, gene findings and annotation, metabolic reconstruction) and data delivery;
- 16S rRNA and other target genes sequencing by Next generation sequencing using Illumina technology (Metabarcoding)
- Bioinformatics analyses: micro-organisms (bacteria, yeast, fungi) identification

Key Organisms

Nicotiana tabacum and benthamiana

Arabidopis thaliana

Plant Cell Suspension Cultures

Zea mays

Agrobacterium temefaciens

E. coli strains

Industrial Applications

- Production for recombinant protein and peptide for:
- Diagnostic kits in the food and health sectors
- Enzymes for a wide range of industrial uses
- Peptides for cosmetic industry
- Drugs and inhibitors discovery/optimization for human health
- Pathogen detection and characterization for food quality and safety (food industry) and animal health (livestock industry)