The course contents address various aspects of molecular biology including heterologous protein expression techniques: from genomic DNA to protein isolation. Extraction of genes and classical and modern high efficiency gene cloning techniques. Protein over-expression techniques in bacterial organisms. Protein purification procedures and metalloprotein reconstitution.
No specific textbooks are available. Book chapters and review articles on specific subjects will be provided to the students.
Learning Objectives
Knowledge and application of the main molecular biology techniques for heterologous expression and purification of recombinant proteins.
The ultimate goal of teaching is to make the student capable of expressing and purifying a protein in order to be studied by spectroscopic analysis.
Prerequisites
Courses required: none
Courses recommended: none
Teaching Methods
25 hours of lectures and 35 hours of practical experiences in laboratory.
Further information
Attendance at the laboratory is compulsory.
Type of Assessment
The exam consists of an oral interview to verify the knowledge of the principal experimental methodologies deployed in the course. It will also be verified the understanding and critical analysis capabilities of the activities carried out during laboratory exercises. During the interview, the student's ability to apply the acquired knowledge to some practical problems proposed by the teacher will be verified.
Course program
DNA - Plasmid - Mutagenesis
Polymerase Chain Reaction - PCR amplification of genes
Gel Electrophoresis - PCR product isolation by gel
Restricting Enzymes
Expression vectors
Preparation of the cloning vector: digestion, dephosphorylation, gel vector isolation.
Ligase Reaction - Transformation
Protein expression in bacterial organisms and cell cultures
Cell breakdown - Nucleic acid removal. Salting in. Salting out.
Dialysis. Ultrafiltration
Methods to Determine Protein Concentration
Chromatographic purification techniques: chromatography by anion exchange, by affinity, by molecular exclusion
Protein electrophoresis
Techniques for the reconstitution of a metalloprotein
Post-translational modifications of proteins
Gateway System
Cell-free
Production of proteins with different types of isotopic markings: 13C, 15N and 2H.
Circular dichroism