This course introduces students to the processes of recombinant protein production in Escherichia coli. Specifically, the aim of the course is for students to acquire the theoretical principles and practical skills needed to express, purify, and characterize recombinant products. Recombinant proteins are biotechnological products widely used as laboratory reagents as well as diagnostics and therapeutics in biomedicine. Therefore, knowledge of the processes and mastery of the techniques used in their production are of paramount importance to a Biotechnologist.

CLASSROOM LESSONS

1. Spectrophotometric protein assays

1.1 Absorbance at 280 nm

1.2 Bradford method

1.3 Lowry method

1.4 BCA method

2. The Escherichia coli expression system

2.1 Advantages of recombinant protein expression

2.2 Features of the E. coli expression system

2.3 Expression vectors

2.3.1 Multicloning site and codon usage

2.3.2 Replication origin and plasmid copy number

2.3.3 Strong and regulatable promoters (lac, trp, PL,T7)

2.3.4 Antibiotic resistance genes and recessive markers

2.3.5 Ribosome Binding Site

3. Strategies to prevent recombinant protein degradation in vivo

3.1 Bacterial proteases and their cellular localization

3.2 Manipulation of culture conditions

3.3 Host engineering

3.4 Product engineering

4. Strategies to prevent the formation of inclusion bodies

4.1 Protein folding and inclusion body formation

4.2 Manipulation of culture conditions

4.3 Host engineering

4.3.1 "Oxidant" strains

4.3.2 Co-expression with molecular chaperones

4.4 Product engineering

4.4.1 Secretion into the periplasm

4.4.2 Fusion proteins (MBP, GST, SUMO)

4.5 Fusion tags

5. Recovery of bioactive recombinant proteins from inclusion bodies

5.1 Protein solubilization

5.2 Refolding in vitro

6. Electrophoretic techniques for protein analysis

6.1 Physical aspects of the electrophoretic separation

6.2 Polyacrilamide gels

6.3 Ionic properties of aminoacids

6.4 SDS-PAGE

6.5 Discontinuous gel system

6.6 Gel staining methods

6.7 Western immunoblotting

6.8 Native PAGE

6.9 Electrophoretic mobility shift assay (EMSA)

7. Bacterial lysate preparation and initial fractionation

7.1 Cryoconservation of the biomass

7.2 Cell disruption

7.3 Composition of the protein extraction buffer

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 Protein assay at 280 nm and setting up of a protein standard curve for the Bradford assay

11.2 Bacterial inoculation, growth, induction of recombinant protein expression

11.3 Cell disruption, protein extraction and initial fractionation

11.4 SDS-PAGE analysis of recombinant protein expression and solubility

11.5 Protein purification by immobilized metal affinity chromatography

11.6 SDS-PAGE analysis of the chromatographic fractions

11.7 Set up of an anzymatic assay for fusion partner removal

11.8 western immunoblotting analysis of the recombinant product

11.9 Titration of an antibody against a recombinant antigen

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

Teaching

Classroom lectures (3 CFU) on the principles of techniques and strategies used to express recombinant products

Practical lectures in the laboratory/computer room (5 CFU) on testing expression, solubility, purification and characterization of a recombinant protein with analysis and processing of data and writing them up in graphical form. "Problem solving" activities will be proposed during the lectures.

video lectures (in English with Italian subtitles) for in-depth study or review from JoVE platform with self-assessment quizzes (delivered through direct link on University Moodle platform)

Attendance

To be eligible to take the exam, it is mandatory to have attended at least two-thirds of the laboratory classes, as well as to have uploaded to blended e-learning platform, prior to the closing of the lists, the paper described in "assessment mode."

In order to attend the labs, it is mandatory to have taken and passed the final test of the online courses "General Safety Training for Workers" and "Safety in the Chemical and Biological Laboratory."

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 must load on blended e-learning a pdf file containing all the results obtained during the laboratory sessions, presented as figures/graphs (specific instructions 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.

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