Expanded Genetic Code approaches to introduce post-translational modification into proteins of interest

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

Acetylation-Specific Interference by Anti-Histone H3K9ac Intrabody Results in Precise Modulation of Gene Expression

Among Histone post-translational modifications (PTMs), lysine acetylation plays a pivotal role in the epigenetic regulation of gene expression, mediated by chromatin modifying enzymes. Due to their activity in physiology and pathology, several chemical compounds have been developed to inhibit the function of these proteins. However, the pleiotropy of these classes of proteins represents a weakness of epigenetic drugs. Ideally, a new generation of epigenetic drugs should target with molecular precision individual acetylated lysines on the target protein. We exploit a PTM-directed interference, based on an intrabody (scFv-58F) that selectively binds acetylated lysine 9 of histone H3 (H3K9ac), to test the hypothesis that targeting H3K9ac yields more specific effects than inhibiting the corresponding HAT enzyme that installs that PTM. In yeast scFv-58F modulates, gene expression in a more specific way, compared to two well-established HAT inhibitors. This PTM-specific interference modulated expression of genes involved in ribosome biogenesis and function. In mammalian cells, the scFv-58F induces exclusive changes in the H3K9ac-dependent expression of specific genes. These results suggest the H3K9ac-specific intrabody as the founder of a new class of molecules to directly target histone PTMs, inverting the paradigm from inhibiting the writer enzyme to acting on the PTM

Selection and characterization of human scFvs targeting the SARS-CoV-2 nucleocapsid protein isolated from antibody libraries of COVID-19 patients

Abstract In 2019, the novel SARS-CoV-2 coronavirus emerged in China, causing the pneumonia named COVID-19. At the beginning, all research efforts were focused on the spike (S) glycoprotein. However, it became evident that the nucleocapsid (N) protein is pivotal in viral replication, genome packaging and evasion of the immune system, is highly immunogenic, which makes it another compelling target for antibody development alongside the spike protein. This study focused on the construction of single chain fragments variable (scFvs) libraries from SARS-CoV-2-infected patients to establish a valuable, immortalized and extensive antibodies source. We used the Intracellular Antibody Capture Technology to select a panel of scFvs against the SARS-CoV-2 N protein. The whole panel of scFv was expressed and characterized both as intrabodies and recombinant proteins. ScFvs were then divided into 2 subgroups: those that exhibited high binding activity to N protein when expressed in yeast or in mammalian cells as intrabodies, and those purified as recombinant proteins, displaying affinity for recombinant N protein in the nanomolar range. This panel of scFvs against the N protein represents a novel platform for research and potential diagnostic applications

Selection and characterization of human scFvs targeting the SARS-CoV-2 nucleocapsid protein isolated from antibody libraries of COVID-19 patients

<p>The Data Set is a collection of the Fasta of sequencing of 6 human library of scFvs (VH-linker VL) obtain from the IgM repertoire and of 6 human libraries of scFvs (VH-linker VL) obtain from the IgG_IgA repertoire of 6 patients that recover from COVID19.</p> <p>Each of the 12 libraries (6 for the IgM and 6 for the IaG/IgA repertoire) were sequenced by ION TORRENT technology by Genomnia. For each scFv library an independently VH and a VL Ion Torrent library was constructed as follows. VH and VL were amplified using primers (VH_fus_F- VH_fus_R and VL_fus_F - VL_fus_R_BXX) having, at the 3’end, the region presents in the pLinker220 plasmid flanking the VH or VL and, at the 5’ end, the Ion Torrent adaptors. In this step, Unique Molecular Identifiers (UMI) consisting of 13 degenerate bases are also introduced in the reverse primer used to amplify the VH and VH regions.  The amplified VH and VL were then subjected to a second PCR using the primers A_fus – trP1_fus to build the fusion library. Sequencing was performed on an Ion Torrent platform (IonS5) with 400bp chemistry on a 530 chip. Both types of sequencing libraries were constructed by Genomnia. Each VH library was individually sequenced to a 400bp read on a 530 chip, yielding approximately 20-25 million reads per sample. The VL libraries, on the other hand, were barcoded and sequenced in a pool of three libraries from different patients, aiming to obtain 6-10 million reads per library. All sequencing runs were of good quality, with an average of 73% of the reads mapping to immunoglobulin genes used for clonotype identification (i.e. less than 30% polyclonal reads) and less than 20% low-quality sequences</p> <p> </p> <p><span> The correspondence between the library number (#) and the # of the patient in the sequencing  files name is as follows:</span></p> <table> <tbody> <tr> <td> <p><strong><span>Library #</span></strong></p> </td> <td> <p><strong><span>Patient # in the file name</span></strong></p> </td> </tr> <tr> <td> <p><span>lib 1 </span></p> </td> <td> <p><span>pz24</span></p> </td> </tr> <tr> <td> <p><span>lib 2 </span></p> </td> <td> <p><span>pz26</span></p> </td> </tr> <tr> <td> <p><span>lib 3 </span></p> </td> <td> <p><span>pz27</span></p> </td> </tr> <tr> <td> <p><span>lib 4 </span></p> </td> <td> <p><span>pz39</span></p> </td> </tr> <tr> <td> <p><span>lib 5 </span></p> </td> <td> <p><span>pz42</span></p> </td> </tr> <tr> <td> <p><span>lib 6 </span></p> </td> <td> <p><span>pz44</span></p> </td> </tr> </tbody> </table> <p><span> </span></p> <p> </p> <p>The correspondence between the library number (#) and the file name for each of the VH and  sequencing is as follows:</p> <table> <tbody> <tr> <td> <p><strong><span>Library #</span></strong></p> </td> <td> <p><strong><span>File name VH Sequences</span></strong></p> </td> </tr> <tr> <td> <p><span>lib 1 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-163-30pM_24_IgM_VH_414__REANALYSIS_488_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 1 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-162-30pM_24_IgG_IgA_VH_407_469_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 2 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-157-30pM_pz26_VH_IgM_404_REANALYSIS_460_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 2 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-158-30pM_pz26_VH_IgG_IgA_403_458_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 3 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-161-30pM_27_IgM_VH_408_465_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 3 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-164-30pM_27_IgG_IgA_VH_413_482_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 4 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_II_Auto_user_S5-00513-159-30pM_pz39_IgM_VH_406_461_rawtf.fa</span></p> <p><span>PANANTICOVID_I_Auto_user_S5-00513-159-30pM_pz39_IgM_VH_406_REANALYSIS_477_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 4 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-160-30pM_pz39_IgG_IgA_VH_405_463_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 5 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_II_Auto_user_S5-00513-166-30pM_pz42_VH_IgM_415_486_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 5 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-167-30pM_42_IgG_IgA_VH_418_489_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 6 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-168-30pM_44_IgM_VH_417_491_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 6 IgG_IgA<span>  </span>VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-169-30pM_pz44_IgG_IgA_VH_420_493_rawtf.fa</span></p> </td> </tr> </tbody> </table&gt