|teoria||5||I sem.||Paola Dominici|
|laboratorio||1||I sem.||Paola Dominici|
Course Description: The course introduces fundamental tools and techniques currently used to engineer protein molecules. Covers the methods used to analyze the impact of these alterations on different protein functions with specific emphasis on enzymatic catalysis. Uses case studies to reinforce the concepts covered, as well as to demonstrate the wide impact of protein engineering research.
Course Significance: Protein engineering is an interdisciplinary field where engineering principles and practices are utilized to generate molecules with novel properties.
The applications of this technology can be found in diverse areas including: drug discovery,
industrial chemical synthesis, transgenic plant research, and nanotechnology.
1. Protein Engineering
• General concepts
• Which targets can be addressed?
• How to choose between rational design and evolutive methods
2. Generation of mutant libraries
• Random mutagenesis, DNA shuffling, Error-prone PCR
• Saturation mutagenesis, SeSaMmethod et
• Examples for successful applications: Directed evolution of DCase; Directed evolution of alpha amylase
3. Emerging principles in protease-based drug discovery. Aspartate proteases inhibitors: inhibitors of HIV protease.
4. The Tumor Suppressor p53: From Structures to Drug Discovery.
Structure–function–rescue: the diverse nature of common p53 cancer mutants
3. Methods for selection
• Bacterial, phage or ribosome display
• Design of bacterial selection systems
4. Methods for screening
• Classes of enzymes and appropriate assays
• Automatization (devices, materials)
5. Expression systems
• Choice of host organisms, vectors, expression in E.coli,
● Purification of fusion and non fusion proteins
6. Strategies for rational design
Cloning and expression of a selected protein /enzyme in E. coli;
Rational design of a mutant; mutagenesis.
An individual project will be assigned, and a written presentation (power point) will be discussed for the final exam.