B016295 - METALLOPROTEIN EXPRESSION LABORATORY

Italian

This course will focus on selected aspects of molecular biology which include the principles for understanding protein expression techniques including DNA manipulation, sequencing, classical gene and high throughput cloning techniques, protein expression in bacterial organisms and cell-free, protein purification, post translational modifications, reconstitution of metal sites in metalloproteins, and uniform and selective isotope labelling techniques of overexpressed proteins.

T.A. Brown Gene Cloning: an Introduction Ed. Chapman & Hall; Keith Wilson and John Walker Metodologia biochimica, Raffaello Cortina Editore; Ivano Bertini, Harry B. Gray, Edward I. Stiefel, Joan Selverstone Valentine, Biological Inorganic Chemistry Ed. University Science Book

Acquire knowledge and learn the methods required for heterologously expressing metalloproteins in bacterial, eukaryotic and in vitro expression systems. Develop the skills necessary to perform the basic activities of a laboratory expressing heterologous metalloproteins through collective laboratory sessions. The final objective of the course is to enable the student of expressing and purifying a metalloprotein in order to be studied through spectroscopic analysis.

Courses to be used as requirements (required and/or recommended)

Courses required: none

Courses recommended: none

Total number of hours for Lectures (hours): 25
Total number of hours for Laboratory-field practice : 35

Combined with Structure and Reactivity of metalloproteins exam (at least eight annual exam sessions).

The course will deal with the heterologous protein expression techniques: from genomic DNA to the isolation of a protein. The basic procedures of gene cloning will be illustrated. In particular, two main approaches of gene cloning will be described, the first based on restriction enzymes and the second on the Gateway technology. The protein expression and overexpression in bacterial organisms and through cell-free approaches and the purification procedures of proteins expressed in these organisms will be explained. Finally, the main post translational modifications in proteins and the reconstitution of metal sites in heterologuos expressed metalloproteins will be described. The subjects of the course will be dealt with lectures and laboratory sessions.