69348 - Chemical and Molecular Methods for the Study of Proteins

Course Unit Page

Academic Year 2021/2022

Learning outcomes

At the end of the course, the student knows the methods for protein and metallo-protein production and purification, both from the biological sources and from recombinant techniques. He learns the physical principles and the applications of calorimetric, spectroscopic and light scattering techniques, to characterize the conformation and the oligomeric state of proteins in solution, their conformational changes and interactions, which are linked to their function.

In particular the student i) proposes experimental strategies to express and isolate proteins of interest in their native form; ii) knows spectroscopic techniques to study secondary and tertiary structure and the conformational changes; iii) understands light scattering techniques to define protein oligomeric state and hydrodynamic properties; iv) understands the use of calorimetry to define the conformational state and the protein-protein and protein-ligand interactions, including metal ions.

During the lab activities the student expresses and purifies a recombinant metallo-protein and learns how to use programs for analyze calorimetric and light scattering data.

Course contents

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

Protein production (8 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 structural stability (8 hours) - Circular dichroism. Protein folding: thermodynamic and kinetic considerations. Folding forces. Conformational funnel. Study of structural stability by perturbing the structure. Chemical and thermal denaturation followed with circular dichroism, differential scanning fluorimetry, differential scanning calorimetry. Intrinsically disordered proteins.

Biomolecular interactions (6 hours) - Isothermal titration calorimetry: theory and applications. Light scattering to study protein quaternary structure. Microscale thermoforesis.

Analysis of experimental data (4 hours) - Use of software to analyze protein-metal titration data and enzymatic activity obtained by isothermal titration calorimetry. Quantitative interpretation of circular dichroism data. Light scattering interpretation software.

Laboratory experience (30 hours) - Heterologous protein expression in E. coli, purification with chromatographic techniques, purity verification with SDS-PAGE, denaturation and interaction alayses.

 

Readings/Bibliography

Scientific articles will be indicated by the teacher.

Teaching methods

There will be 4 CFU (32 hours) of teaching, including four hours of computer activity in the Bioinformatic lab. The laboratory activity will be of 2 CFU (30 hours) at Belmeloro Laboratory.

Assessment methods

The final assessment can be performed in two different ways, according to the student's choice.

1) Interactive option: the student will attend the lectures and the classroom activities and will revise the information at home. The grade will be determined according to the sum of the scores of these different activities:

- Presentation of a deepening of a topic of the course:7

- Computer data analysis: 2

- Laboratory report (group activity): 3

- Portfolio (to be filled up after every topic explained in class):8

- Test (multiple choice questions): 10

2) Classic option: written exam. Three questions to be answered on  different topics of the course. The lab report will be considered to round up the final grade.

Teaching tools

The literature and teaching slides will be shared through the Moodle platform.

Office hours

See the website of Barbara Zambelli