The course aims to illustrate and convey to students the principles and applications of biochemical techniques for studying the expression, production in recombinant form, purification and characterisation of proteins. Proteins are important targets for pharmacological intervention, but are also widely used as biopharmaceuticals, in the development of diagnostics, and as biocatalysts and technical enzymes in industry. Therefore, learning the methodologies inherent to protein biochemistry and recombinant DNA techniques is an integral part of the training of people with chemical, pharmacological and technological skills.

1.   Introduction to the study of proteins

Recalls on protein structure. Protein folding. Post-translational modifications. Intracellular compartments and protein sorting

2.     In vitro, ex-vivo and in vivo models for studying proteins

2.1 Cell cultures: basic instrumentation; culture media, propagation and storage; primary cell cultures and immortalised and tumour cell lines

2.2 Organoids and organoids, transgenic mice and knock-out mice (outline)

 3.     Gene expression analysis and manipulation techniques

3.1 DNA and RNA extraction techniques, PCR, Real Time PCR, microarrays.

3.2 Overview of gene expression manipulation techniques (antisense, decoy, siRNA, CRISP-Cas9)

 4.     Cell lysis and tissue disruption methods

4.1 Physical, mechanical and chemical methods

4.2 Extraction buffer formulation

4.3 Centrifugation techniques and cell fractionation.

5.     Protein assay methods

Acid hydrolysis and spectrophotometric methods (280 nm, Bradford, Lowry, BCA)

 6.   Protein analysis techniques

6.1 Electrophoretic techniques

6.1.1 Physical aspects of electrophoretic separation. Polyacrylamide gel. SDS-PAGE. Continuous and discontinuous gel/pad system. Staining methods. 

6.1.2 PAGE under native conditions.

6.1.3 Isoelectrofocalisation and two-dimensional separations.

6.2 Immunochemical techniques.

6.2.1 Mono- and polyclonal antibody production, immunization strategies, immunoglobulin purification techniques

6.2.2 Western immunoblotting: blocking; direct and indirect detection; antibody marking and detection methods

6.2.3 ELISA techniques (direct, simple and sandwich indirect, competitive)

6.3 Mass spectrometry-based proteomic approaches

6.4 Enzyme assays

6.4.1 Recollections of enzyme kinetics; experimental assessment of the steady state kinetics: measurement of the initial rate, single substrate enzyme reactions, two-substrate enzyme reactions, enzyme inhibitors

6.4.2 Substrate dosage.

7.     Recombinant DNA technology for protein production

7.1 Purposes of recombinant protein expression

7.2 Escherichia coli as an expression system

7.2.1 Plasmid cloning and expression vectors, Multiple cloning site and codon usage, Replication origin and plasmid copies, The strong and adjustable promoters (lac promoter, tryptophan, ptac, pL,T7, pBAD), Antibiotic resistance genes and recessive markers, The Ribosome Binding Site.

7.2.2 Strategies to prevent proteolysis in vivo: bacterial proteases and their intracellular localisation, manipulation of fermentation conditions, host engineering, product engineering.

7.2.3 Strategies to prevent the formation of inclusion bodies; manipulating fermentation conditions; engineering the host; engineering the product. Secretion in the periplasm. Fusion proteins (MBP, GST, UB, SUMO). Use of proteases for fusion partner removal and self-tagging peptides. 

8.     Protein purification methods

8.1 Recovery of bioactive proteins from inclusion bodies and biotechnological applications of inclusion bodies.

8.2 Cell extract clarification techniques

8.3 Recall of the main chromatographic techniques.

8.4 Phases and objectives of a purification protocol; development of a specific assay; selection and combination of fractionation techniques; yield and degree of purification.

8.5 Dialysis, ultrafiltration, lyophilisation and storage.

9.  Affinity chromatography applied to the purification of fusion proteins

The matrix, The ligand, The spacer arm. Elution techniques. Biological and structural ligands used for the purification of recombinant proteins.

10.  Bacterial endotoxins: structure and clinical implications.

10.1 Detection techniques for endotoxin contaminants (rabbit test, Limulus Amoebocyte Lysate test, recombinant tests, MAT test)

10.2 Removal techniques for endotoxin contaminants.

 11.  Characteristics of other expression systems

Yeasts, mammalian cells, insect cells, plants: advantages and disadvantages

12.  Examples of recombinant proteins used as biopharmaceuticals and industrial enzymes

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D1-Knowledge and ability to understand. The student will have to demonstrate knowledge of biochemical and molecular methodologies used for the analysis of expression, isolation, identification and characterisation of proteins, including recombinant proteins. He/she should also know and understand the potential of proteins in pharmacology, diagnostics and industry.
D2-Ability to apply knowledge and understanding. The student should be able to select the most appropriate techniques to solve specific problems in different contexts ranging from basic research to industrial production.
D3-Autonomy of judgement. The student must demonstrate the ability to argue methodological choices on the basis of the limitations associated with the various techniques studied, proposing alternative solutions or integrative analyses.
D4-Communicative skills. The student must be able to express him/herself in appropriate language using the technical terminology of the sector. He should also be able to make references to related topics in a logical manner.
D5-Learning ability. The student should be able to read autonomously and critically the literature of the sector, deepening the aspects of interest. The student should 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

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Teaching

Lectures using slides and video tutorials from the JoVe platform to illustrate the practical part
 

Attendance

none

Course books

M. Duranti. Introduzione allo studio delle proteine. Zanichelli 

M. C. Bonaccorsi di Patti, R. Contestabile, M. L. Di Salvo. Metodologie Biochimiche - Espressione, purificazione e caratterizzazione delle proteine. Zanichelli

other texts

M. Maccarone. Metodologie biochimiche e biomolecolari. Strumenti e tecniche per il laboratorio del nuovo millennio. Zanichelli

K. Wilson, J. Walzer. Metodologia Biochimica, le Bioscienze e le Biotecnologie in Laboratorio. Raffaello Cortina Editore.

Assessment

Oral interview. The student will be asked at least three questions to verify the level of knowledge and depth of the topics of the course programme and the ability to reason developed by the student in dealing with specific problems relating to protein biochemistry.
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.

Teaching

In order to give non-attending students the opportunity to compensate with self-study what is carried out during the lectures, the following materials referring to the same contents of the syllabus are provided in order to promote their full understanding:

selection of videos on some of the topics covered accompanied by self-assessment quizzes. Links to the videos and quizzes will be provided on the Moodle platform together with slides of the lessons

Attendance

none

Course books

M. Duranti. Introduzione allo studio delle proteine. Zanichelli 

M. C. Bonaccorsi di Patti, R. Contestabile, M. L. Di Salvo. Metodologie Biochimiche - Espressione, purificazione e caratterizzazione delle proteine. Zanichelli

other texts

M. Maccarone. Metodologie biochimiche e biomolecolari. Strumenti e tecniche per il laboratorio del nuovo millennio. Zanichelli

K. Wilson, J. Walzer. Metodologia Biochimica, le Bioscienze e le Biotecnologie in Laboratorio. Raffaello Cortina Editore.

Assessment

Oral interview. The student will be asked at least three questions to verify the level of knowledge and depth of the topics of the course programme and the ability to reason developed by the student in dealing with specific problems relating to protein biochemistry.
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.