• intrabody
• expanded genetic code
• biologia sintetica
• biologia molecolare
• anticorpi
• lievito
• neurobiologia

In the past few years, the ability to incorporate unnatural amino acids (UAAs) into proteins at defined sites has begun to have a direct impact on the ability of scientists to study biological processes that are difficult or impossible to address by more classical methods. One of the most powerful approaches for incorporating UAAs site-specifically into protein expressed in cells is the genetic code expansion. In this approach, an aminoacyl-tRNA synthetase and a tRNA are used to specifically insert the unnatural amino acid during mRNA translation, in response to an amber stop codon (UAG) placed at a user-defined site in a gene of interest. This has allowed new biological insights into protein conformational changes, protein interactions, elementary processes in signal transduction and, the role of post-translational modifications (PTMs).
In this thesis, we employed the orthogonal synthetase/tRNAPylCUA (PylRS/tRNACUAPyl ) pairs from Methanosarcina barkeri (Mb) and M. mazei (Mm), that enables the incorporation of acetyl-Lysine into protein produced in L40 strain of S. cerevisiae. This pairs has emerged as a particularly versatile and orthogonal system for genetic code expansion in E. coli, yeast Saccharomyces cerevisiae and mammalian cells.
However, current methods for incorporating UAAs in yeast are based on transient transfection, that lead to very heterogeneous expression, limiting the ability to couple precise perturbation, which may be effected by UAAs mutagenesis, to global proteomic measurements. To overcome this limitation, stable yeast lines, expressing synthetase and tRNA from an integrated locus, will be created for acetyl-Lysine incorporation (“acetylator” yeast).
Using this method, several recombinant proteins bearing a post translation modification, in our case acetylation, at a define site can be produced. Lysine acetyla¬tion is a key post-translational modification in regulating chromatin structure and function, resulting of epigenetic phe¬nomena that regulates diverse biological processes, from metabolism to signalling. In this thesis, acetyl-lysine will be inserted into recombinant H3 protein and microtubule associated protein TAU. In the first case, the acetylation of H3 will provide new insights into the role of this PTM in regulating chromatin structure and function; in the second case it would be useful in deciphering the biological and pathological role of different TAU acetylations. To address these questions the newly created acetylator yeast strain will be employ to produce H3 or acetylated TAU proteins that will be used to select intracellular antibodies (intrabodies) that specifically recognize the PTM. This will be done using the Intracellular Antibody Capture (IAC) technology, a yeast two hybrid based technique that allows screening of intrabodies libraries to specific targets (in our case the acetylated H3 or TAU protein).
The newly selected intrabodies will then be used as tools to understand/dissect the role of the specific PTM on the whole protein function.
Given the growing list of amino acid that can be incorporated using PylRS/tRNAs variants, the natural follow up of this work will be the creation of new stable yeast lines for the introduction of a wide range of other constitutive PTMs, such as phosphorylation or methylation.