UCL - Ingénierie des protéines en enzymologie

[BBMC2105]

[36h + 18h] 5 credits

Teacher(s): Patrice Soumillion

Language: French

Level: Second

Aims
Main themes
Content and teaching methods
Other information (prerequisite, evaluation (assessment methods), course materials recommended readings, ...)

Aims

The objective of the course is to deepen the understanding of the properties of natural proteins and to introduce the student to the field of protein engineering that allows artificial evolution towards new properties. The student will learn some of the advanced investigation methods in enzymology and protein science as well as the theoretical and practical notions that are related to protein stability and folding. Then, he will get to know the different engineering strategies currently used as well as the associated biotechnologies. With the help of recent case studies describing directed, random, combinatorial and in silico approaches, the student will understand the actual limitations and difficulties of protein engineering but also its possibilities and future challenges. He/she will also study the properties obtained by engineering and compare them with the natural properties of proteins. The notion of directed evolution will be introduced and the description of some examples will aim at acquiring a vision of the artificial mechanisms of evolution in comparison with our knowledge of natural mechanisms.

Main themes

The main topics of the course will be :1. Protein science1.1. Protein stability, folding and dynamics:- thermodynamics of protein stability and folding (theory and methods of investigation)- reversible and irreversible denaturation - in vivo protein folding (folding pathways, disulfide bonds formation, proline isomerisation, protein chaperones, conformationnal diseases)- spectroscopic methods (FRET, BRET, single molecule spectroscopy) 1.2. Enzymology- practical aspects of enzymology (assays, enzyme inactivation, experimental design)- estimation of rate constants (experimental and analytical problems)- mathematical simulation and optimisation (derivation of rate equations, numerical integration, analysis of experimental data)- multi-substrate reactions and multi-enzyme systems- isotope exchange and isotope effects- fast reactions (pre-steady-state kinetics, active site titration, burst kinetics, experimental techniques)2. Protein engineering- techniques for mutagenesis and combination of mutations (directed mutagenesis, error prone PCR, incorporation of degenerated oligonucleotides, DNA shuffling)- screening libraries (characteristics of screening assays, high throughput screening, examples)- in vivo selection (principle, examples)- in vitro selection (phage display and similar technologies, compartmentalisation)- engineering new protein-ligand interactions- enzyme engineering (specificity, regulation, catalysis)- chemical modification of proteins in vitro and in vivo- protein engineering in silico

Content and teaching methods

The course will begin with a short reminder of protein biochemistry. With the help of examples chosen in recent scientific literature, about 30 hours will then be devoted to three main themes:

(1) Modern methods for creating protein variants:
- site directed and random mutagenesis, recombinogenesis (DNA shuffling), synthetic oligonucleotides incorporation;
- non natural amino acids incorporation
- synthetic or semi-synthetic peptidic ligation (use of inteins)
- genetic fusions with or without susbequent chemical modifications in vitro or in vivo

(2) Screenings and Selections:
- colorimetric, fluorimetric, microbiologic and analytic assays
- high throughput screenings
- in vivo selection
- in vitro selection (phage display and compartmentalisation)

(3) New fields of applications of engineered proteins:
- fine structure-function relationships studies
- new tools for molecular and cellular biology
- biocatalysis
- biomedicine
- biotechnology

After the lectures, students will work individually on research articles. During one month, weekly meetings of questions and answers will be organized for discussing different aspects of the articles (state of the art, strategic choices, experimental methodology, rigour in data treatment and interpretation). Each student will finally present his article to the rest of the group by giving a 30 min lecture.

Other information (prerequisite, evaluation (assessment methods), course materials recommended readings, ...)

Precursory courses:
- Protein biochemistry (e.g. BBMC2101 Structural and Functional Biochemistry)
- Basics in molecular biology (e.g. BBMC2102 Integrated Molecular and Cellular Biology)

Evaluation: Presentation of research articles
Support: PowerPoint slides