LABORATORY OF RECOMBINANT PROTEINS
- Overview
- Assessment methods
- Learning objectives
- Contents
- Full programme
- Bibliography
- Teaching methods
- Contacts/Info
The student who attends this course will be asked to apply the knowledge acquired in the core curriculum of the First Cycle Degree: Biochemistry, in particular solid knowledge regarding the structure and properties of proteins are required; Molecular Biology, with particular reference to recombinant DNA techniques (manipulation, amplification, cloning) but also to gene expression regulation mechanisms; Microbiology, as regards the distinctive features of the microorganisms used as heterologous hosts for the production of recombinant proteins and their maintenance in culture. In order for the student to be able to carry out the experimental work during the planned laboratory activities, the knowledge and practical skills acquired during the course of Biochemical Methodologies are required.
The exam is organized in a written test concerning the modules of “Protein Engineering” and “Laboratory of Recombinant Proteins” (four open questions for each module). Laboratory activities will be evaluated through a written scientific report of the experimental procedures and results. The final score is given in 30ies: the examination is successful if a score 18/30 is gained in each module and in the laboratory activities report.
This course of Protein Engineering and Laboratory Recombinant Proteins is fundamental to the preparation of a student in Industrial and Molecular Biotechnology. The aim of the course is to explain the molecular mechanisms at the basis of the structure-function relationships of proteins and the experimental approaches to modulate the protein functionality and to evolve a desired function or structure. The course is also aimed to discuss the most updated knowledge/skills related to the production of recombinant proteins. It therefore provides the theoretical tools for the design and production in different heterologous systems of protein variants.
In particular, the Protein Engineering module aims to provide the knowledge of the different techniques in the biotechnological application of protein engineering: design of protein structures from scratch, rational design for the design of new functions, molecular modeling and docking, mutagenesis (site-directed and random) as well as methods through which it is possible to isolate the optimized protein variants in a given property.
The Laboratory of Recombinant Proteins module is designed to provide an overview of the different expression systems used in the production of recombinant proteins for different applications (industrial or pharmaceutical). For each system the most recent methods for the preparation of expression constructs, the regulation of protein expression levels and the optimization of production yields will be exposed.
Students will be encouraged to evaluate the advantages and limitations of the various approaches used to modify proteins properties and those related to the use of different systems of heterologous expression. Case studies will be also discussed. The course includes laboratory activities during which the theoretical contents will be applied experimentally. Students are expected to acquire decision-making skills in a "protein engineering" project, from the design phase to the production of recombinant protein variants one, as well as to be able to face and solve the various problems related to the process.
Learning outcomes can be summarized as follows.
Cognitive skills:
- acquisition of fundamental elements at molecular level concerning protein structure-function relationships;
- acquired the required information (theoretical and experimental) to carry out the process of engineering of a protein function/structure.
Learning abilities:
- ability to read, understand and criticize a scientific text on the production, characterization and engineering of recombinant proteins (in English);
- ability to use these knowledge to evaluate the aims and/or the results of a research project both on the qualitative and quantitative point of view.
Practical and subject specific skills:
- ability to apply the knowledge in the field of biochemistry and molecular biology to a protein engineering project;
- ability to propose an analytical problem-solving approach.
Communication skills:
- ability to identify and express relevant information;
- demonstrate effective communication skills by practicing, reading, writing and speaking clearly;
- demonstrate the ability to resume and present the scientific information.
Module “Laboratory of Recombinant Proteins” –Silvia Sacchi: 6 CFU – 24 h class, 48 hours laboratory activities
- Introduction recombinant proteins production
- Cloning techniques and expression vectors
- "Cell free" systems for protein expression
- Expression in prokaryotic systems
- Expression in E. coli
- E. coli fermentation
- Strategies to increase the expression of proteins in soluble form
- Expression in yeast - Pichia, Saccharomyces etc
- Expression in insect and mammalian cells
- Criteria for the choice of the best expression host and final discussion
Laboratory activities (see the detailed program)
Module “Laboratory of Recombinant Proteins” –Silvia Sacchi: 6 CFU – 24 h class, 48 hours laboratory activities
- Introduction recombinant proteins production: general information on heterologous expression systems (currently used and under development) and their application potential.
- Cloning techniques and expression vectors.
- "Cell free" systems for protein expression: in vitro transcription and/or translation applied to the production of recombinant proteins - advantages, limits and potential fields of application.
- Expression in prokaryotic systems: expression of recombinant proteins in bacteria (Gram negative and Gram positive) - advantages and limits of use.
- Expression in E. coli.
- E. coli fermentation: process optimization with genetic and physiological tools. Scaling up problems, from the laboratory scale to the pilot and production plants.
- Strategies to increase the expression of proteins in soluble form: strategies to be adopted to minimize the formation of insoluble protein aggregates (inclusion bodies; inclusion bodies with "reservoir" of recombinant proteins - methods for purification, solubilization and refolding.
- Expression in yeast - Pichia, Saccharomyces etc: eukaryotic expression systems, most commonly used vectors and promoters (constitutive and inducible), expression methods (stable or transient, in the cytoplasm or in the extracellular medium following secretion); advantages and limitations of the expression system.
- Expression in insect and mammalian cells: eukaryotic expression systems, vectors and promoters, methods of expression, culture conditions related to the different cell lines and to the production needs, advantages and limitations of the expression system.
- Criteria for the choice of the best expression host and final discussion: open discussion on the course’s contents.
Note: 8 h class will be dedicated to the exposition of subjects of primary importance to be prepared for laboratory activities.
Laboratory activities
- Activity 1 - Preparation of EP-PCR reactions in different amplification conditions;
- Activity 2 - Screening of a library of enzymatic variants through a colorimetric assay.
- Activity 3 - Expression trials of a protein variant, medium preparation, inoculation and monitoring of the fermentation process (measurement of optical density and pH).
- Activity 4 - Expression trials of a protein variant, induction and and cells collection.
- Activity 5 - Expression trials of a protein variant, cell extracts preparation, assay for the determination of the total protein concentration and the enzymatic activity; discussion of the results.
- Activity 6 - Expression trials of the pLG72 protein (produced as inclusion bodies), use of different recombinant E. coli strains in order to improve the protein solubility.
- Activity 7 - Expression trials of pLG72 protein, fermentation monitoring of the fermentation process (measurement of optical density and pH), induction and collection.
- Activity 8 – Expression trials of pLG72 protein, preparation of cell extracts, assay of the total protein concentration, SDS-PAGE and Western blot analysis.
- Activity 9 - Expression trials of pLG72 protein, discussion of results.
For the Laboratory of Recombinant Proteins module a reference text is not available; in addition to the slides of the lessons, further material to complete the preparation (scientific papers and reviews) can be downloaded from the e-learning website. Additional material may be also requested.
Module “Protein Engineering” (L. Pollegioni) 6 CFU - 48 h class
Module “Laboratory of Recombinant Proteins” (S. Sacchi) 6 CFU - 24 h class 48 h laboratory activities.
Students can meet with the professor in his office by previous email appointment.