RECOMBINANT PROTEINS
- Overview
- Assessment methods
- Learning objectives
- Contents
- Full programme
- Delivery method
- Teaching methods
- Contacts/Info
The student who attends this course will be asked to apply the knowledge acquired in the core cur-ric-ulum of the First Cycle Degree: Biochemistry; Molecular Biology, (and in particular recombinant DNA techniques such as amplification and cloning, but also gene expression regulation mecha-nisms); and Microbiology.
In order for the student to be able to carry out the experimental work during the planned laborato-ry activities, the knowledge and practical skills acquired during the course of Biochemical Methodol-ogies are required.
Verification of learning will take place through a written test aimed at assessing the learning achievements and the understanding of the problems discussed during classes as well as the ac-quired knowledge. The student will be given 4 open questions (each evaluated up to 8 points).
The laboratory activity will be evaluated by an oral exposition of the obtained results. Group of stu-dents who carried out the same set of experiments will be asked to prepare a PowerPoint presen-tation reporting about procedures and the experimental outcomes, which they will then discuss with the lecturer and their colleagues
The outcome of the exam will be out of thirty: the exam is considered passed with a mark of at least 18/30. The final mark will be the arithmetic average of the outcome of the two tests (the written exam and the oral exposition of the practical activities), 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 problems related to the expression of recombinant proteins;
3. the ability to apply the acquired knowledge to set up an expression strategy in one or more suita-ble heterologous systems;
4. the ability to propose solutions to specific problems by using the tools and strategies discussed during teaching;
5. clarity of the exposed concepts and the use of appropriate scientific terminology.
Educational objectives
The selection of the best heterologous production system is of utmost importance to obtain high-quality and functional recombinant proteins for different applications, ranging from biomedical research to industrial processes. For this reason, this course is designed to provide an overview of the different and currently exploited expression systems and to allow students acquiring 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.
The course is among those proposed for the Bio-based Industry curriculum and provide students with a solid preparation in the design of construct and the identification of heterologous systems for the production of recombinant proteins, to be used as biotechnological tools in the biomedical (as therapeutic agents) and industrial (as components of bioconversion and biocatalysis production pro-cesses) field, in line with the educational objectives proposed by the CdS. Students will be encour-aged to evaluate the advantages and limitations related to the use of the different heterologous expression systems. Case studies will be also discussed.
The course includes laboratory activities during which the theoretical contents will be applied exper-imentally. Students are expected to learn what kind of problems can arise during the expression of heterologous proteins in the discussed systems and the strategies to adopt for avoiding them and possibly optimizing the yield of the recombinant product.
Learning Outcomes
At the end of the course, students will be able to:
• develop strategies for the production of recombinant proteins based on their properties, critically choosing the most appropriate heterologous expression system;
• apply the theoretical notions to the resolution of problems connected with the production of re-combinant proteins, in order to maximize production yields;
• evaluate, extract and synthesize the information acquired, when relevant for a specific project or case study;
• communicate and discuss effectively the notions learned both orally and in writing, using an appropriate scientific language.
Students will acquire decision-making skills in a "recombinant protein expression project", from the design to the production phase, being able to face and solve the various critical issues related to the process.
6 ECTS divided into 32 hours of lectures (4 ECTS), 24 hours of practical laboratory activities (2 ECTS)
The topics covered during the course are the following:
Lessons
- Introduction to the production of recombinant proteins
- Cloning techniques and expression vectors
- Expression in E. coli
- Expression in alternative prokaryotic systems (gram negative and gram positive bacteria)
- Actinomycetes as a valuable expression system (seminar by Dr. Francesca Berini)
- Attractive expression systems: extremophiles
- Expression in eukaryotic hosts: yeasts (methylotrophic and non-methylotrophic)
- Expression in in eukaryotic hosts: insect cells – the baculovirus system
- Expression in in eukaryotic hosts: mammalian cell lines
- "Cell free" systems for the expression of recombinant proteins
- Hints to further systems of expression and final considerations
Laboratory
- Application of directed evolution techniques - Error prone PCR
- Application of directed evolution techniques - screening of enzymatic variants
- Expression trials for production yields optimization - effect of the composition of the growth medi-um
- Expression tests for the optimization of the production yields of recombinant proteins – using dif-ferent expression strain to improve the protein solubility
- Critical discussion of the results
6 ECTS divided into 32 hours of lectures (4 ECTS), 24 hours of practical laboratory activities (2 ECTS)
The topics covered during the course are the following:
Lessons
- Introduction to the production of recombinant proteins
- Cloning techniques and expression vectors
- Expression in E. coli
- Expression in alternative prokaryotic systems (gram negative and gram positive bacteria)
- Actinomycetes as a valuable expression system (seminar by Dr. Francesca Berini)
- Attractive expression systems: extremophiles
- Expression in eukaryotic hosts: yeasts (methylotrophic and non-methylotrophic)
- Expression in in eukaryotic hosts: insect cells – the baculovirus system
- Expression in in eukaryotic hosts: mammalian cell lines
- "Cell free" systems for the expression of recombinant proteins
- Hints to further systems of expression and final considerations
Laboratory
- Application of directed evolution techniques - Error prone PCR
- Application of directed evolution techniques - screening of enzymatic variants
- Expression trials for production yields optimization - effect of the composition of the growth medi-um
- Expression tests for the optimization of the production yields of recombinant proteins – using dif-ferent expression strain to improve the protein solubility
- Critical discussion of the results
The module consists of 32 hours of lectures and 24 hours of practical activities in the lab. Each lesson will be carried out by treating a specific topic (from cloning techniques, to individual heterologous expression systems) focusing on specific problems related to the production of recombinant proteins and the strategies to possibly avoid or solve them. PowerPoint presentations with the slides discussed during lectures, will be available in advance on the e-learning platform. Papers reporting case studies will be discussed and provided as didactic material.
For practical activities (6 practice 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 scheduled activities).
The lecturer is always available to receive students, preferably by appointment (via requests to the email address silvia.sacchi@uninsubria.it). Prof. Sacchi is also available for in-depth or clarification meetings for groups of students on the topics covered by the course, that will be scheduled in the same way.