LABORATORY OF RECOMBINANT PROTEINS
Lecturer: Prof. Silvia Sacchi
6 ECTS divided into 24 hours of lectures (3 ECTS), 48 laboratory hours (3 ECTS)
Lessons:
- Introduction to the production of recombinant proteins
- Cloning techniques and expression vectors
- "Cell free" systems for the expression of proteins
- 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
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 pro-teins - effect of the composition of the growth medium
- Preparation of expression tests for the optimization of the production yields of recombinant pro-teins - use of various E. coli strains to improve the yield of the product in soluble form
- Comparison and critical discussion of the results
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.
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.
The module consists of 24 hours of lectures and 48 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).
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.