Ateneo - UPO

The course introduction illustrates the application of biochemical methods to different fields in the bio-medical research area. This section also briefly recaps the chemical, physical and functional properties of nucleic acids and proteins, which can be exploited for the isolation, the production, the analysis and the manipulation of these fundamental macromolecules. The following part of the course is structured in two main teaching modules. The first section describes the recombinant DNA techniques and the systems that can be used (or adapted) to produce recombinant proteins, including the demonstration of the use of publicly accessible databases for the analysis and comparison of nucleotide and amino acid sequences. The second section describes the currently available technology, instrumentation, materials and methods for i) the purification of proteins and macromolecular complexes (which can be obtained from natural sources or expressed in recombinant form), ii) their biochemical characterisation, and iii) their modification or functional improvement to serve specific biochemical tasks.

Stoppini, M e Bellotti, V “Biochimica Applicata” Ed. EdiSES ISBN 9788879597135; Bonaccorsi di Patti, MC, et al. “Metodologie Biochimiche” Ed. CEA ISBN 978-8808-18329-3.
Selected full-papers and reviews aimed at triggering the student's further independent study on the taught subjects. The slideshows supporting the lectures will be made available to the students (in the absence of copyright issues). Exam test examples will be made available. All these contents will be published on "DIR" at the end of each module (nucleic acid purification and analysis; protein purification and analysis; macromolecular complexes analysis).

The Course "Applied Biochemistry" describes the theoretical and practical aspects, and the impact, of the study of biological macromolecules in different fields, from medical and life sciences to bio-analytical disciplines. The course aims to equip the student with a solid background for her/his future study and research activities, focused on the description of the structure and function of proteins and the application of the obtained information. To this end, classical and advanced methods for the production, purification, manipulation and biochemical characterisation of macromolecules will be illustrated. Virtual research projects will be assigned to small groups of students, aiming at stimulating student's attitude to an independent and critical thinking and to work in team.

A basic knowledge of the biochemistry of nucleic acids and proteins, as well as of DNA replication, transcription and protein synthesis processes, in prokaryotic and lower eukaryotic species, is required.

Standard lessons.
Three/four 2-hr lessons will be used to allow the students to present and discuss/defend their "virtual" projects on a peer-to-peer basis.

Audio/video material (in English) would be used during the lessons. Exercises on the use of databases will be scheduled (computer room).

Written exam.
The first exercise will focus on the steps required to design and construct a vector for the expression of a recombinant protein, and on the biochemical/functional analysis of the encoded protein (max. 8 pt). The second exercise will consist in defining a strategy for the over-expression (in a heterologous system), the purification and the biochemical and/or functional characterisation of a recombinant protein (max. 10 pt). The short assay may relate to a technical topic (e.g. "Principles and applications of affinity chromatography") or to a broader subject (e.g. "Analysis of macro-molecular interactions by using biophysical methods) (max. 12 pt). The test must be carried out in its entirety, obtaining at least the following scores: 1st exercise 5/8; 2nd exercise: 5/10; open question: 6/12. Up to 3 additional points will be assigned to the members of each group that will present and discuss the “virtual project”.

The recombinant DNA technology applied to the expression of proteins. The Polymerase Chain Reaction: principles, equipment and design of primers. Electrophoresis on agarose gel for DNA analysis: principles and instruments. Sub-cloning vectors: general characteristics. Plasmid vectors for the expression of recombinant proteins in bacteria. Other vectors for the generation of recombinant DNA molecules. Transformation of competent bacteria. Genomic DNA purification. The production of cDNA libraries. Restriction endonuclease and ligases: general characteristics, physiological role and use in the recombinant DNA technology. Purification of DNA fragments from agarose matrices. Purification of plasmid DNA from bacterial cultures. PCR-based site-directed mutagenesis: principles and examples. DNA sequencing: the "Sanger's" method and its automation. Analysis of nucleic acids by hybridisation techniques: principles and applications. DNA probes labelling techniques. The expression of recombinant proteins in other heterologous systems; in vitro coupled transcription-translation. Systems for the inducible expression of recombinant proteins in E. coli. The baculovirus/insect cell system (the Bac-to-Bac system in detail). The Tet-ON and Tet-OFF systems for the inducible expression of recombinant proteins in mammalian cells. The yeast Pichia pastoris as a recombinant protein expression system. Working with proteins: the golden rules. Cellular disruption (lysis) by osmotic shock, mechanical stress and use of detergents: principles, limits and applicability of the methods. Cellular lysis by ultra-sonication: principles and applications. Centrifugation of biological samples: principles and instrumentation. Cell fractionation by differential centrifugation. Quantification of proteins in a sample. Protein electrophoresis: principles and instrumentation. The electrophoretic separation of proteins and analysis by SDS-PAGE. Protein analysis by isoelectric-focusing (IEF): principles and applications. 2D Electrophoresis; "total" or "group-specific" staining methods of proteins separated by electrophoresis. Protein detection by immunoblot: western blot and dot-blot. Immunological tests: general principles. ELISA, RIA and competitive assays. The lateral flow immuno-chromatography applied to disposable devices. Introduction to Liquid Chromatography techniques (LC) used in purification of macromolecules. Affinity chromatography: matrices modified with group-specific and mono-specific ligands. Chromatographic approaches for the isolation of recombinant "tagged" proteins. Ion exchange chromatography (IEC). Hydrophobic interaction chromatography (HIC). Size exclusion chromatography (SEC) and its application to the analysis of protein-protein interactions and oligomeric state. Methods for concentration of proteins in a sample. Analysis of amino acid composition and primary protein structure. Determination of the N- and C-terminal residues. Edman's Degradation. Biochemical study of macromolecular interactions. The "classic" two-hybrid yeast system (2HYS) for confirming protein interactions and as an interaction cloning approach. Other "N-hybrid" artificial systems for the study of macromolecular interactions, at specific localisation within the cell. The phenomenon of surface plasmon resonance (SPR) applied to the quantitative, in real-time study of interactions between macromolecular partners and of protein / small molecule association: principles and instrumentation. Procedures / methods for biosensor chip functionalization. The in vitro and in vivo analysis of macromolecular complexes dynamics based on the FRET phenomenon: principles and applications.

Upon successful completion of the course, the students will have acquired knowledge and understanding of the taught subjects, and gained skills to autonomously deal with and solve new and/or complex problems on the studied topics. In details, they will know and understand a) the methods, the techniques and the experimental protocols for the production and purification of proteins and nucleic acids to be used in biotechnology and diagnostic field and b) the methods, techniques and experimental protocols for the manipulation and the biochemical/functional characterisation of proteins, nucleic acids and their complexes. The students will be able to apply this knowledge a) to set up an experimental protocol for the expression and / or purification of proteins; b) to adapt an already published procedure to specific cases in the field of the biochemical and functional analysis of biological macromolecules; c) to develop new functional assays, by properly choosing the experimental method and the equipment for conducting the experiments. Finally, the students are expected to build up the capability to query the correct sources and databases, and to extract the significant information, developing the learning skills necessary to undertake further study on the subjects covered by the course, with a high degree of autonomy. The students will be able to use the appropriate terminology to communicate the results of "problem solving" activities. Overall, these capabilities will serve as the basis for i) a more informed understanding of other disciplines that the students will encounter in their ensuing studies and ii) for a more autonomous and conscious conduction of the laboratory activities aimed at their experimental thesis data collection and analysis.