Ateneo - UPO

The “Introductory notes” of the “Applied Biochemistry” course illustrate the application of biochemical methods to different fields in bio-medical and biotechnology research areas; this section also recaps the chemical, physical and functional properties of nucleic acids and proteins that can be exploited for the isolation, the production, the analysis and the manipulation of these fundamental biological macromolecules. The following part is structured in two main teaching modules: the first one describes the recombinant DNA techniques and the systems that can be used to produce recombinant proteins, including the use of publicly accessible databases for comparing nucleotide and amino acid sequences; the second section describes some currently available technologies for the purification and analysis of macromolecular complexes (from natural sources or expressed in recombinant form.

-Stoppini, M e Bellotti, V “Biochimica Applicata” Ed. EdiSES ISBN: 9788879597135
- Bonaccorsi di Patti, MC, et al. “Metodologie Biochimiche” Ed. CEA ISBN: 9788808183293
-Amaldi, F et al. “Tecniche e metodi per la biologia molecolare“ ISBN: 9788808920348
- Further selected Reviews and scientific articles (full papers), suggested by the Lecturer.
The pdf files of the presentations used during the lectures will be made available to the students at the end of each macro-section (e.g. purification of proteins); they should be considered as a guide for the detailed study of the same topics on the suggested textbooks.

The "FA0419-Applied Biochemistry" course describes the theoretical and practical aspects of the study of biological macromolecules, and the impact of the discipline in different fields, ranging from life sciences to analytical applications. The course aims to equip the student with a solid background for her/his future research activities in the protein science area, and the exploitation of the gained knowledge also in the study of other disciplines. To this end, advanced methods for the production, purification, manipulation and biochemical characterisation of macromolecules will be illustrated.

A basic knowledge of the biochemistry of proteins and nucleic acids and of the processes responsible of the biological information transmission is required.

Standard lectures. Audio/video materials (in English) will be used during the lectures. Scientific papers will be discussed. Final test simulations and training sessions on problem solving will be regularly scheduled.

Students with physical disabilities, Learning Disabilities or Special Education Needs can request specific services and tools via the Staff Sviluppo e Coordinamento Carriere e Servizi alle Studentesse e agli Studenti, consulting the University webpage: https://www.uniupo.it/en/services/services-students-physical-or-learning-disabilities Students with disabilities, learning disabilities or special education needs, once they have contacted the University Staff, can refer to the tutor in charge of the course to define the examination modalities, concerning academic aspects.

Written exam. The first exercise asks the students to design a vector for the expression of a recombinant protein (max. 7 pt); the second exercise consists in the definition of a strategy for the over-expression, the purification and the functional characterisation of a recombinant protein (max. 10 pt); the short assay may relate to a technical subject (e. g. principles and applications of affinity chromatography) or to a broader topic (e. g. analysis of macro-molecular interactions) (max. 13 pt). To pass the test students must obtain at least the following scores: 1st exercise 5/7; 2nd exercise: 6/10; short assay: 8/13.

Summary of the characteristics and of the biological macromolecules that can be exploited for their purification and analysis, and of the cellular processes involved in nucleic acid and protein synthesis/modification. The recombinant DNA technology applied to the expression of proteins. The Polymerase Chain Reaction: principles, equipment, primer pairs and specific protocol design. 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. Purification of plasmid DNA from bacterial cultures. Genomic DNA purification. The production of cDNA libraries. Restriction endonuclease and ligases: general characteristics and use in the recombinant DNA technology. Purification of DNA fragments from agarose matrices. Analysis of nucleic acids by hybridization techniques: principles and applications. The expression of recombinant proteins in heterologous systems: an overview. Systems for the inducible expression of recombinant proteins in E. coli. The baculovirus/insect cell system. In vitro coupled transcription-translation.
DNA sequencing techniques.
Working with proteins: the golden rules. Cellular lysis by osmotic shock, mechanical stress and use of detergents: principles, limits and relevance of the methods. Cellular lysis by ultrasonication: principles and applications. Centrifugation of biological samples: instrumentation. Cell fractionation by differential centrifugation. Quantification of proteins in a sample. Protein electrophoresis: principles and instrumentation. The electrophoretic separation of proteins by SDS-PAGE. Protein analysis by isoelectric focusing (IEF): principles and applications. 2D Electrophoresis. Total or group-specific staining methods to visualize proteins upon gel electrophoresis. Protein detection by immunoblot. Immunological tests: general principles. ELISA, RIA and competitive assays. The lateral flow immunochromatographic assay: examples of disposable devices for rapid testing. Introduction to Liquid Chromatography techniques (LC) for protein purification. Affinity chromatography (AC): matrices modified with group-specific and mono-specific ligands. AC approaches for the isolation of tagged recombinant proteins. Ion exchange chromatography (IEC). Hydrophobic interaction chromatography (HIC). Size exclusion chromatography (SEC) and its application to the analysis of protein-protein interactions and protein oligomeric state in solution. Methods to concentrate proteins in a sample. Considerations on the strategies to be adopted for setting up assays for enzyme characterization and for monitoring the steps during a protein purification procedure. The study of interactions between macromolecules: an introduction. Pulldown and co-immunopurification. The in vitro and in vivo analysis of macromolecular complexes dynamics based on the FRET phenomenon: principles and applications.
The two-hybrid yeast system (2HYS) to confirming protein interactions and as an interaction cloning approach.

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 the methods and techniques for the production and purification of proteins and nucleic acids to be used in biotechnology and diagnostic field and for the biochemical/functional characterization of proteins, nucleic acids and macromolecular complexes. The students will be able to apply this knowledge to adapt an already published procedure to a specific case, in order to perform a biochemical and/or functional analysis of a given biological macromolecule. 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 theoretical basis i) for an informed understanding of other disciplines that the students will encounter in the following of their curriculum, and ii) to perform lab activities aimed at collecting/analysing biochemical and cell biology data.