The course introduces students to the processes of recombinant protein production in Escherichia coli. In particular, the purpose of the course is to let the students to acquire the theoretical principles, and the practical skills necessary to express, purify and characterize recombinant products. Recombinant proteins are biotechnology products widely used as laboratory reagents as well as diagnostics and therapeutics in biomedicine. Thus, the knowledge of the processes and the mastery of the techniques used for their production are of fundamental importance for a Biotechnologist.

CLASSROOM LESSONS

1. Electrophoretic techniques for protein analysis
1.1 Ionic properties of aminoacids
1.2 Physical aspects of the electrophoretic separation
1.3 Polyacrilamide gels
1.4 SDS-PAGE
1.5 Discontinuous gel system
1.6 Gel staining methods
1.7 Western immunoblotting
1.8 Native PAGE
1.9 Electrophoretic mobility shift assay (EMSA)
2. Spectrophotometric protein assays
2.1 Absorbance at 280 nm
2.2 Bradford method
2.3 Lowry method
3. The Escherichia coli expression system
3.1 Advantages of recombinant protein expression
3.2 Features of the E. coli expression system
3.3 Expression vectors
3.3.1 Multicloning site and codon usage
3.3.2 Replication origin and plasmid copy number
3.3.3 Strong and regulatable promoters (lac, trp, PL,T7)
3.3.4 Antibiotic resistance genes and recessive markers
3.3.5 Ribosome Binding Site
4. Strategies to prevent recombinant protein degradation in vivo
4.1 Bacterial proteases and their cellular localization
4.2 Manipulation of culture conditions
4.3 Host engineering
4.4 Product engineering
5. Strategies to prevent the formation of inclusion bodies
5.1 Protein folding and inclusion body formation
5.2 Manipulation of culture conditions
5.3 Host engineering
5.3.1 "Oxidant" strains
5.3.2 Co-expression with molecular chaperones
5.4 Product engineering
5.4.1 Secretion into the periplasm
5.4.2 Fusion proteins (MBP, GST, SUMO)
5.5 Fusion tags
6. Bacterial lysate preparation and initial fractionation
6.1 Cryoconservation of the biomass
6.2 Cell disruption
6.3 Composition of the protein extraction buffer
7. Recovery of bioactive recombinant proteins from inclusion bodies
7.1 Protein solubilization
7.2 Refolding in vitro
8. Designing a purification scheme
8.1 Lysate clarification
8.2 Key steps in a purification protocol
8.3 Selection of a specific assay for the protein of interest
8.4 Selection and logical combination of purification techniques
8.5 % yield and purification enhancement
9. Purification of fusion proteins by affinity chromatography
9.1 The matrix
9.2 The ligand
9.3 The spacer arm
9.4 Elution techniques
10. Characterization of the protein product
10.1 Bacterial endotoxins: structure and clinical implications
10.2 Techniques for the detection of endotoxin contaminants
10.2.1 Rabbit pyrogen test
10.2.2 Limulus Amoebocyte Lysate (LAL) test (gel clot, chromogenic, turbidimetric, recombinant)
10.2.3 Test of cytokine release
10.3 Methods for endotoxin removal
10.4 Mono- and polyclonal antibodies
10.5 Immunization techniques
10.6 Immunoglobulin purification methods
10.7 ELISA
10.7.1 direct ELISA
10.7.2 indirect ELISA
10.7.3 competitive ELISA

LABORATORY LESSONS

11. Expression and purification of a recombinant protein: from the theoretical principles to the laboratory practice
11.1 Preparation of the stock solutions for SDS-PAGE
11.2 Setting up of a protein standard curve for the Bradford assay
11.3 Preparation of the culture medium for bacterial growth, buffers for bacterial lysis and extraction of soluble and insoluble proteins
11.4 Bacterial inoculation, growth, induction of recombinant protein expression
11.5 SDS-PAGE analysis of recombinant protein expression
11.6 Cell disruption, protein extraction and initial fractionation
11.7 SDS-PAGE of the soluble and insoluble fraction
11.8 Protein purification by affinity chromatography
11.9 SDS-PAGE analysis of the chromatographic fractions
11.10 Cleavage of the fusion partner
11.11 SDS-PAGE and Western Immunoblot analysis of the recombinant protein
11.12 LAL test, gel clot
11.13Titration of an antibody against a recombinant antigen by indirect ELISA

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D1-Knowledge and comprehension. Students will get familiar with the most widely used laboratory techniques and experimental strategies to express, purify and characterize recombinant proteins. In particular, they will learn about the advantages and disadvantages of using E. coli as expression system and how to face the latter by manipulating the fermentation conditions or by host/ product genetic engineering. They will know how to store the biomass, prepare a bacterial extract, design a purification protocol. They will gain knowledge with the principles and possible applications of the employed laboratory techniques. They will learn about the most advanced immunological techniques and endotoxin detection methods used to characterize recombinant products for diagnostic/therapeutic applications.

D2-Ability to apply knowledge and comprehension. Students will be able to follow, under the supervision of specialized personnel, a recombinant protein production process, being able to understand all the steps: from the expression, to the purification and characterization of the final product. They will understand and execute procedures described on a provided experimental protocol and set up autonomously a simple protocol upon definition of the objectives and goals of the experiment.

D3-Autonomy of judgment. Students will be able to register, graphically present data and critically comment on the results obtained in the laboratory. They will be able to identify problems related to the production process and propose solutions.

D4-Comunication skills. Student will be able to describe a production process and the techniques employed, as well as comment on the experimental results using an appropriate language.

D5-Learning ability. The student will have the basics to read autonomously and critically the literature of the sector, deepening the aspects of interest. They will be able to ask questions and provide answers.

The teaching material prepared by the lecturer in addition to recommended textbooks (such as for instance slides, lecture notes, exercises, bibliography) and communications from the lecturer specific to the course can be found inside the Moodle platform › blended.uniurb.it

Lectures (20 h, including laboratory sessions) given by Sabrina Dominici PhD, Senior R&D Researcher (Diatheva srl, Cartoceto PU)

Teaching

Lectures and laboratory lessons. The schedule for laboratory activities will be established at the beginning of the course

Attendance

Students must attend at least 2/3 of the classroom lessons and 2/3 of the laboratory lessons.

To attend laboratory classes it is compulsory to have followed and passed the final test of the online courses “General Safety Training for Workers” and “Safety in the Chemical and Biological Laboratory” (Safety referent, Prof. Annarita Mastrogiacomo).

In order to carry out the teaching activities properly, it is highly recommended to have passed the exam or at least followed the courses of “Biotechnology Laboratory I” and “Biotechnology Laboratory II”. It is also important to have acquired the contents of the courses of Biochemistry and Molecular Biology.

Course books

B.R. Glick, J.J. Pasternak, Biotecnologia Molecolare, Principi e Applicazioni del DNA Ricombinante, Zanichelli.
K. Wilson, J. Walzer, Metodologia Biochimica, le Bioscienze e le Biotecnologie in Laboratorio, Raffaello Cortina Editore.
M. C. Bonaccorsi di Patti, R. Contestabile, M. L. Di Salvo, METODOLOGIE BIOCHIMICHE - Principi e tecniche per l'espressione, la purificazione e la caratterizzazione delle proteine. Casa Editrice Ambrosiana.
R. K. Scopes, Protein purification, principles and practice, Springer-Verlag.

other material (slides, protocols and reviews) will be provided during the course

Assessment

Oral Interview. Students will have at least three questions regarding theoretical notions with specific references to laboratory practice (techniques and procedures). At least one of the questions will specifically address the content of the lessons in the lab. To this end, the students will have to provide the teacher with the elaboration in the form of figure (s) of the collected experimental data (the modalities will be determined during the course and specific instructions on the nature of the work will be provided during the lessons). During the exam, the teacher will ask to describe and comment critically what is shown in one of the figures according to the scheme: purpose, procedure, result, comment.

The final score will be determined by: the level of mastery of theoretical notions and the ability to apply them to concrete examples, the level of articulation and relevance of responses, language ownership, including the use of appropriate technical terminology, exposure capacity .

Disability and Specific Learning Disorders (SLD)

Students who have registered their disability certification or SLD certification with the Inclusion and Right to Study Office can request to use conceptual maps (for keywords) during exams.

To this end, it is necessary to send the maps, two weeks before the exam date, to the course instructor, who will verify their compliance with the university guidelines and may request modifications.

For laboratory lessons students will be divided into two / four groups. Subdivision of groups and subgroups will be established at the beginning of the course.

The student can request to sit the final exam in English with an alternative bibliography.