Unit PROTEIN BIOTECHNOLOGY
- Course
- Molecular and industrial biotechnology
- Study-unit Code
- A003308
- Curriculum
- In all curricula
- Teacher
- Lorena Urbanelli
- Teachers
-
- Lorena Urbanelli
- Hours
- 47 ore - Lorena Urbanelli
- CFU
- 6
- Course Regulation
- Coorte 2024
- Offered
- 2024/25
- Learning activities
- Caratterizzante
- Area
- Discipline biologiche
- Academic discipline
- BIO/10
- Type of study-unit
- Obbligatorio (Required)
- Type of learning activities
- Attività formativa monodisciplinare
- Language of instruction
- Italian
- Contents
- The structural levels of proteins and protein folding. The catalytic function of proteins: kinetics, mechanisms, inhibition, regulation. The production of recombinant proteins in prokaryotes, simple eukaryotes (yeasts) and mammals. The production and optimization of recombinant proteins in the medical and industrial sectors.
- Reference texts
- Petsko-Ringe Struttura e funzione delle proteine, Zanichelli
- Educational objectives
- The main objective of the course is to provide students with a good understanding of the structural and functional characteristics of proteins, their methods of production, purification and optimization, as well as their potential applications in the various biotechnological sectors. The knowledge acquired will allow the student to understand the different modes of action and critically evaluate the parameters that can influence their activity in order to be able to predict possible applications in the various biotechnological sectors, with particular regard to the industrial sector. The practical exercises will provide the student with the necessary tools to apply the theoretical knowledge acquired in order to solve problems and design experiments.
- Prerequisites
- To understand and profitably attend the course, it is useful for the student to have a good knowledge of basic elements of general and inorganic chemistry, organic chemistry and biochemistry
- Teaching methods
- The course is organized as follows:
- lectures in the classroom related to the topics covered by the program. Lessons will be carried out with the aid of slide projections and films;
- final seminar of each student with the presentation of a case study of the production of a protein;
- practical activity in the laboratory. The students will be divided into groups of 3-4 students per group and will personally carry out the analyses foreseen in the course programme. The practical part will be preceded by an introduction in the classroom. During the exercises the students will be assisted by expert and qualified laboratory personnel. - Other information
- For the calendar of teaching activities and the start and end dates of the lessons, please consult the degree course website: http://www.dcbb.unipg.it/laurea-magistrale-in-biotecnologie-molecolari-e-industriali
- Learning verification modality
- The exam includes a 15-minute seminar test which consists in the presentation of a case study about the production of a protein and an oral test which consists in a discussion relating to the topics covered during the course. The duration of the exam may vary depending on the progress of the test itself, with an average duration of about 30-40 minutes in total. The oral exam aims to evaluate the level of knowledge and understanding achieved by the student regarding the topics covered. Furthermore, this interview will make it possible to verify the student's ability to communicate with language properties and clarity of exposition what has been learned
- Extended program
- Part 1. From sequence to structure and function of proteins.
Amino acids: classification and peptide bond, phi and psi rotation angles. Weak ties. The family of helical structures (alpha-helix, collagen, polyproline), the beta sheet, the beta fold. The cis-trans isomerization of proline. Levinthal's paradox and the folding funnel. The concept of dominance and motive. The interaction between interfaces and the quaternary structure. The functional and structural information of the primary structure. Homology modeling and Alphafold. Reversible and irreversible post-translational modifications. Membrane proteins: integral, peripheral and amphipathic. The synthesis of membrane proteins: the SRP system and the role of the ER. Glycosylation in N and O.
Part 2. The catalytic function of proteins.
Catalyzed and non-catalyzed reactions. Activation energy, transition state, binding energy. The kinetics of Michaelis and Menten. The meaning of Vmax and Km: the example of isoenzymes. Enzymes that do not respect Michaelis and Menten kinetics: ordered, random, ping-pong bimolecular reactions. The concept of Kcat. The dependence of the reaction rate on temperature and pH. Enzyme assays. The classification of enzymes. Catalytic mechanisms: acid-base catalysis, covalent, from metal ions. The example of chymotrypsin. Irreversible inhibitors: group-specific reagents, substrate and transition state analogues, suicide inhibitors. Reversible inhibitors: competitive, incompetitive, mixed and their recognition from the Lineweaver-Burk graph. The inhibition constant, IC50 and EC50. The reversible mechanisms of regulation of enzymatic activity.
Part 3. The production of recombinant proteins
Protein production by recombinant DNA technology: host choice. Advantages and disadvantages of protein production in bacteria. The plasmid vectors and the expression cassette. Fusion proteins: advantages and disadvantages. Optimization Strategies In E.coli. Yeasts: plasmid, replicative and integrating vectors. Plasmid expression vectors in mammalian cells and DNA entry into the cell: the concept of non-viral gene delivery. Viral gene delivery: retroviruses and packaging lines. First, second and third generation lentiviral vectors. First, second and third generation (gutless) adenoviral vectors. Optimization strategies: rational design, guided evolution, design from scratch. Site-specific and random mutagenesis.
Part 4. The production of recombinant proteins in the medical and industrial sectors.
Optimization of insulin, yeast hepatitis B vaccine, tissue plasminogen activator. Industrially optimized proteins: amylase, pectinase, Cellulase, Laccase and Lipase.
Part 5. Individual seminar activity regarding the production and/or optimization of a protein of interest in the biomedical or industrial sector
Part 6. Practical laboratory activities consisting in the purification of an enzyme - Obiettivi Agenda 2030 per lo sviluppo sostenibile
- The course provides skills to deal with the research, development and production of biotechnological products in the field of health and industrial production processes