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 curriculum of the First Cycle Degree: Biochemistry; Molecular Biology, (and in particular recombinant DNA techniques such as amplification and cloning, but also gene expression regulation mechanisms); and Microbiology. 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 achievements and the understanding of the problems discussed during classes as well as the acquired 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 students who carried out the same set of experiments will be asked to prepare a PowerPoint presentation 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 suitable 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 Recombinant proteins have become central components in a wide range of biotechnological applications, from therapeutic monoclonal antibodies and vaccine antigens to industrial enzymes, food-grade proteins, and innovative biomaterials. The ability to produce high-quality recombinant proteins efficiently and economically is crucial across sectors including biomedical, industrial, pharmaceutical, and agrifood. This course provides an in-depth overview of recombinant protein production, with a focus on heterologous gene expression in microbial, mammalian, insect, and emerging cell-free systems. Par-ticular attention is given to the strategic selection of the expression host, the design of expression constructs, and optimization strategies to maximize yield, solubility, activity, and post-translational modifications. We will also explore recent trends in host system engineering, such as synthetic biology approaches, the development of novel chassis organisms, and the integration of sustainable bioproduction methods in line with the needs of the bio-based economy. This course, part of the Bio-based Industry curriculum, provides students with a strong foundation in the design and implementation of heterologous expression strategies for the production of recom-binant proteins. It also includes practical lab sessions, which allow students to consolidate theoretical concepts while gaining hands-on experience in the experimental production and optimization of recombinant proteins. Learning Outcomes By the end of the course, students will be able to: • Design appropriate strategies for recombinant protein production based on protein properties and intended applications, critically selecting the most suitable heterologous expression system; • Optimize gene expression and process parameters to improve yield, solubility, and activity of recombinant proteins, applying troubleshooting approaches to solve common production challenges; • Interpret and synthesize scientific data relevant to protein expression workflows, and apply this knowledge to project design, case studies, and problem-solving tasks; • Communicate technical and scientific concepts effectively, both orally and in written form, using appropriate scientific language and formats; • Demonstrate independent decision-making in the planning and execution of a recombinant protein expression project, from construct design to production and basic downstream analysis; • Evaluate emerging technologies and expression platforms in light of sustainability, scalability, and specific functional requirements.
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 1. Introduction to Recombinant Protein Production; 2. Cloning Techniques and Expression Vectors; elements, codon optimization, fusion tags. 3. Prokaryotic expression systems; From E. coli to engineered bacteria; 4. Yeasts and Protozoa as eukaryotic hosts; 5. Insect and mammalian cell systems; 6. Cell-free systems and synthetic platforms; 7. Strategies for difficult-to-express proteins; 8. Downstream Considerations: yield, folding, and activity; 9. Emerging trends and future directions. 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 medium - Expression tests for the optimization of the production yields of recombinant proteins – use of different expression strain to improve the protein solubility - Critical discussion of the results
Lessons 1. Introduction to Recombinant Protein Production. Overview of applications, production platforms, and technological trends. 2. Cloning Techniques and Expression Vectors. DNA assembly, regulatory elements, codon optimization, fusion tags. 3. Prokaryotic expression systems. From E. coli to engineered bacteria Including Gram-positive, Gram-negative, and extremophilic bacteria. 4. Yeasts and Protozoa as eukaryotic Hosts. Pichia pastoris, Saccharomyces cerevisiae, and LEXSY systems. 5. Insect and mammalian cell systems; Baculovirus expression, HEK293, CHO, and glycoengineering. 6. Cell-free systems and synthetic platforms; For rapid screening, toxic proteins, and membrane protein synthesis. 7. Strategies for difficult-to-express proteins. Comparative overview of advanced hosts and approaches tailored for unstable, membrane-associated, or heavily modified proteins. 8. Downstream Considerations: yield, folding, and activity; Solubility, aggregation, proteolysis, and protein quality control. 9. Emerging trends and future directions. Biofoundries, machine learning for construct design, and sustainable bioproduction.
The course 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 pro-teins 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.