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

1.1 Recalls on protein structure.

1.2 Protein folding.

1.3 Post-translational modifications.

1.4 Intracellular compartments and protein sorting.

1.5 Protein degradation

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 Primary cell cultures, immortalized and tumor cell lines, organoids and spheroids

2.3 Transgenic mice, knock-in mice, knock-out mice, and conditional knock-out mice (Cre-Lox system)

3. Methods of cell lysis, tissue disruption, cell fractionation

3.1 Physical, mechanical and chemical methods.

3.2 Extraction buffer formulation

3.3 Centrifugal techniques and cell fractionation.

4 Protein assay methods

4.1 UV/VIS absorption spectroscopy

4.2 Spectrophotometric methods for protein assay (280 nm, Bradford, Lowry, BCA)

4.3 Construction of BSA calibration line by Bradford method

4.4 Fluorescence spectroscopy

4.5 Spectrofluorimetric methods for protein assay

4.6 Methods based on amino acid analysis

5. Techniques for gene expression analysis

5.1 RNA extraction techniques and quantification

5.2  Real Time PCR

5.3 Microarray

6. Electrophoretic techniques

6.1 Physical aspects of electrophoretic separation. 

6.2 Polyacrylamide gel

6.3 SDS-PAGE. Continuous and discontinuous type gel/tampon system.

6.4 Staining methods

6.5 PAGE under native conditions

6.6 Isoelectrofocusing and two-dimensional separations

6.7 Agarose gels for DNA and RNA analysis

7. Immunochemical Techniques

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

7.2 Western immunoblotting: blocking; direct and indirect detection; antibody labeling and detection methods.

7.3 Immonochromatography

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

8.     Recombinant DNA technology for protein production

8.1 Purposes of recombinant protein expression

8.2 Escherichia coli as an expression system

8.3 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.

9. Strategies to prevent proteolysis in vivo

9.1 bacterial proteases and their intracellular localisation, manipulation of fermentation conditions, host engineering, product engineering.

10. Strategies to prevent the formation of inclusion bodies

10.1 inclusion bodies formation

10.2  manipulating fermentation conditions

10.3 engineering the host 8coexpression with molecular chaperons, oxidant strains)

10.4 engineering the product. Secretion in the periplasm. Fusion proteins (MBP, GST, UB, SUMO). Use of proteases for fusion partner removal and self-tagging peptides. 

11.     Protein purification methods

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

11.2 Cell extract clarification techniques

11.3 The main chromatographic techniques.

11.4 Affinity chromatography applied to the purification of fusion proteins. Biological and structural ligands used for the purification of recombinant proteins.

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

11.6 Dialysis, ultrafiltration, lyophilisation and storage.  

12.  Bacterial endotoxins: structure and clinical implications.

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

12.2 Removal techniques for endotoxin contaminants.

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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

Written exam in which students will be asked to explain three of the four topics proposed by the lecturer. The questions are designed to test the level of knowledge and depth of the topics in the course syllabus as well as the ability to select the most appropriate techniques for dealing with specific problems pertaining to protein biochemistry.
Each answer will be assigned from 0 to 10 points. The final grade will be determined by summing the total score expressed in thirtieths.
The following will contribute to determining the grade: the level of mastery of the theoretical concepts, the level of articulation and relevance of the answers, the property of language, including the use of appropriate technical terminology, and the ability to expose.
The test is considered passed if the grade reported in the written exam is at least 18/30.
Students who have scored at least 18/30 in the written test and also wish to take the oral examination may contact the lecturer for the integrative interview. The date of the oral exam will be communicated by the lecturer via blended.

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 the topics covered. Links to the videos will be provided on the Moodle platform together with the 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

Written exam in which students will be asked to explain three of the four topics proposed by the lecturer. The questions are designed to test the level of knowledge and depth of the topics in the course syllabus as well as the ability to select the most appropriate techniques for dealing with specific problems pertaining to protein biochemistry.
Each answer will be assigned from 0 to 10 points. The final grade will be determined by summing the total score expressed in thirtieths.
The following will contribute to determining the grade: the level of mastery of the theoretical concepts, the level of articulation and relevance of the answers, the property of language, including the use of appropriate technical terminology, and the ability to expose.
The test is considered passed if the grade reported in the written exam is at least 18/30.
Students who have scored at least 18/30 in the written test and also wish to take the oral examination may contact the lecturer for the integrative interview. The date of the oral exam will be communicated by the lecturer via blended.

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.