C8508 - Protein Technologies: from Production to Characterization

Learning outcomes

By the end of the course, students will have acquired a solid understanding of the main methods for protein production and purification, both from biological sources and recombinant expression systems. They will understand the chemical and physical principles underlying protein folding and stability and will be familiar with the experimental methods used to characterize these properties. Spectroscopic, calorimetric, and light scattering applications will be described as methods for studying protein folding, oligomeric state, conformational changes, and protein-protein or protein-ligand interactions (including drugs), all closely related to protein function. Students will learn to interpret data obtained from proteins in solution to derive biologically relevant information and evaluate their potential as drug targets. Specifically, by the end of the course, students will be able to: i) design experimental strategies to express and isolate proteins of interest in their native form; ii) apply spectroscopic techniques to study secondary and tertiary structure and conformational changes; iii) use light scattering techniques to assess the oligomeric state and hydrodynamic properties of proteins; iv) understand and apply methods to study protein-protein and protein-ligand interactions. During the laboratory activities, students will purify a recombinant protein and perform its folding and interaction analysis using the techniques covered in the course.

Course contents

Introduction (2 hours): Introduction to the protein study and of the methods to isolate proteins from the biological source or in recombinant form.

Protein expression (6 hours): Cloning methods for recombinant genes. Use of E. coli as expression host. Optimization parameters for protein expression in E. coli. Inclusion bodies. How to increase protein stability: periplasm secretion, chaperones, co-expression. Use of tags and fusion proteins. Optimizing E. coli growth conditions for increasing the protein yield. Protein expression in eukaryotes: yeast cells, insect cells, mammals, plants. "Cell-free" expression systems.

Protein purification (4 hours): Chromatographic techniques for protein separation. Salting in/out. Quality control of purified proteins: protein stability, concentration and purity.

Protein folding and stability (10 hours): Protein folding: thermodynamic and kinetic considerations. Conformational landscape. Folding funnel. Intrinsically disordered proteins. Study of structural stability by perturbing the structure. Chemical and thermal denaturation by differential scanning calorimetry, thermal shift assay, circular dichroism.

Protein interactions (6 hours): Isothermal titration calorimetry. Light scattering to study protein quaternary structure. Protein folding and interactions in the cellular landscape.

Laboratory activities. Protein purification and chatacterization and data analysis (26 hours): Purification of a recombinant protein, spectroscopic analysis and thermal shift assay; analysis of data of thermal shift assay,isothermal titration calorimetry, circular dichroism. Critical interpretation of experimental data and extraction of biologically relevant information.

Readings/Bibliography

Scientific articles will be indicated by the teacher and the didactic material will be shared through Virtuale platform.

Teaching methods

There will be 4 CFU (28 hours) of teaching. The laboratory activity will be of 2 CFU (26 hours) at the Laboratory in via San Donato.

Assessment methods

The assessment consists of a written examination and evaluation of laboratory activities.

The written examination is designed to assess the student's understanding of the theoretical principles and experimental methods presented during the course, as well as their ability to interpret experimental data and design appropriate strategies for protein production, purification and characterization.

The written examination includes:10 multiple-choice questions aimed at assessing knowledge and understanding of the course topics; 2 open-ended questions aimed at assessing the ability to apply concepts, analyze experimental results and discuss methodological approaches.

The written examination is graded on a scale of 30 points.

Participation in laboratory activities and the submission of a laboratory report allow students to obtain up to 2 additional points. The laboratory assessment is based on active participation, data analysis and interpretation, and the quality of the written report.

Students who do not attend the laboratory activities will be required to take an additional oral examination focused on the experimental applications and methodologies covered during the laboratory sessions. The grade obtained in the oral examination will be averaged with the grade of the written examination and will replace the laboratory assessment.

The final score is calculated as the sum of the written examination score (maximum 30 points) and the laboratory assessment score (maximum 2 points), for a maximum total score of 32 points. Final scores above 30 points will be recorded as 30/30 cum laude.

Teaching tools

Teaching materials and supporting learning activities will be made available through the University of Bologna Virtuale platform.

The course will combine lectures with active learning strategies aimed at promoting student engagement and the application of theoretical concepts to real experimental problems. These activities may include:

• Flipped classroom sessions based on the analysis of scientific papers, videos or teaching materials provided before class;
• Group problem-solving activities focused on experimental design, data interpretation and methodological choices in protein production and characterization;
• Interactive quizzes and polls using digital tools (e.g. Wooclap) to assess understanding and provide immediate feedback;
• Guided discussion of case studies from biotechnology, pharmaceutical research and protein-based drug discovery;
• Collaborative analysis of experimental datasets obtained from spectroscopic, calorimetric and light-scattering techniques;
• Laboratory activities involving protein purification, characterization and interpretation of experimental results.

Additional supporting materials, including lecture slides, scientific articles, protocols, datasets and supplementary resources, will be provided through the Virtuale platform.

Students are strongly encouraged to actively participate in all learning activities, as engagement with lectures, discussions, problem-solving exercises and laboratory sessions is expected to enhance the achievement of the intended learning outcomes and the development of discipline-specific competencies.

Office hours

See the website of Barbara Zambelli