LABORATORY OF RECOMBINANT PROTEINS
- Overview
- Assessment methods
- Learning objectives
- Contents
- Bibliography
- Teaching methods
- Contacts/Info
The student who attends this course will be asked to apply the knowledge acquired in the core curric-ulum of the First Cycle Degree: Biochemistry (in particular solid knowledge regarding the structure and properties of proteins are required); Molecular Biology, (recombinant DNA techniques - manipu-lation, amplification, cloning - but also gene expression regulation mechanisms); Microbiology (distinc-tive 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.
Verification of learning will take place through a written test aimed at assessing the learning achieve-ments and therefore the understanding the problems discussed during classes and the acquired knowledge related to the "Protein Engineering" and "Laboratory of Recombinant Proteins" modules. For each module the student will be given 4 open questions (each evaluated up to 8 points).
The laboratory activity will be evaluated by drawing up a detailed scientific report of the experiments carried out by group of students.
The outcome of the exam will be out of thirty: the exam is considered passed with a mark of at least 18/30 in each module and in the laboratory report.
The final mark will be the arithmetic average of the outcome of the three tests, each weighed for the relative ECTS.
The criteria according to which the acquired knowledge and skills will be assessed are:
1. the degree of depth of the subject matter of the question;
2. the critical ability to rework and link the knowledge acquired regarding the theoretical bases of the structure-function relationship in proteins and to the problems related to the expression of recombi-nant proteins;
3. the ability to apply the knowledge acquired to develop a protein engineering project and to design, based on the characteristics of a specific protein of interest, an expression strategy in one or more suitable heterologous systems;
4. the ability to propose solutions to specific problems by using the tools and strategies discussed dur-ing teaching;
5. the clarity of the exposed concepts and the use of appropriate scientific terminology .
This course of Protein Engineering and Recombinant Proteins is fundamental to the preparation of a student in Industrial and Molecular Biotechnology. The teaching is organized into two modules: Pro-tein Engineering and Laboratory of Recombinant Proteins. Together the two modules aim to provide students with a solid preparation in the design and production of recombinant proteins, to be used as biotechnological tools in the biomedical (as therapeutic agents) and industrial (as components of bio-conversion and biocatalysis production processes), in line with the educational objectives proposed by the CdS.
The Laboratory of Recombinant Proteins module is designed to provide an overview of the different expression systems used in the production of recombinant proteins and allow students to acquire in-depth knowledge of the most recent methods for optimizing their use: from the design of expression constructs, to the regulation of the expression levels of the proteins of interest and the optimization of production yields.
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 recombi-nant protein variants one, as well as to be able to face and solve the various problems related to the process.
Lecturer: Prof. Silvia Sacchi
6 ECTS divided into 24 hours of lectures (3 ECTS), 36 laboratory hours (3 ECTS)
Lessons:
- Introduction to the production of recombinant proteins
- Cloning techniques and expression vectors
- Expression in prokaryotic systems
- Expression in E. coli
- Fermentation of E. coli
- Expression in yeast - Pichia, Saccharomyces etc
- Expression in insect and mammalian cells
- Guest selection criteria and final discussion
- "Cell free" systems for the expression of proteins
- Hints to further systems of expression and final considerations
Laboratory:
- Application of a directed evolution technique - Error prone PCR
- Application of a directed evolution technique - screening of enzymatic variants
- Preparation of expression tests for the optimization of the production yields of recombinant proteins - effect of the composition of the growth medium
- Preparation of expression tests for the optimization of the production yields of recombinant proteins - comparison between different expression systems, E. coli vs. CHO
- Critical discussion of the results
A single reference text is not available for the Laboratory of Recombinant Proteins module; in addi-tion to the slides of the lessons, the material useful to complete the preparation (scientific papers and reviews) will be downloadable from the e-learning site. Additional material can be requested from the teacher. Handouts and protocols for the part of practical laboratory exercises will also be available on the e-learning platform.
The module consists of 24 hours of lectures and 36 hours of laboratory. Each lesson will be carried out by treating a specific topic (from cloning techniques, to individual heterologous expression systems, to problems related to the production of recombinant proteins and strategies to solve them), by us-ing powerpoint presentations, available in advance on the e-learning platform. During the lessons, case studies published scientific journals of the field will be discussed (publications will be provided as didactic material).
For practical activities (9 practise of 4 hours each, grouped in two different weeks), students will be divided into small groups. After a general introduction to illustrate the objectives and the practical methods of execution, the students will organize and carry out the experimental activities under the supervision of the lecturer. Attendance to practical activities is mandatory (at least 75% of the sched-uled activities).
A single reference text is not available for the Laboratory of Recombinant Proteins module; in addition to the slides of the lessons, the material useful to complete the preparation (scientific papers and reviews) will be downloadable from the e-learning site. Additional material can be requested from the teacher. Handouts and protocols for the part of practical laboratory exercises will also be available on the e-learning platform.
Reception preferably by appointment (by request via e-mail). Prof. Sacchi replies only to e-mails signed and coming from the domain @ uninsubria.it. We do not respond to requests for confirmation of information already available - for example, request of the exam date.
The lecturer is available for in-depth meetings or discussions for groups of students.