Colarusso, Andrea (2018) Genetic and process engineering for the production of protein therapeutics for the treatment of CNS disorders. [Tesi di dottorato]
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Item Type: | Tesi di dottorato |
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Resource language: | English |
Title: | Genetic and process engineering for the production of protein therapeutics for the treatment of CNS disorders |
Creators: | Creators Email Colarusso, Andrea andrea.colarusso@unina.it |
Date: | 8 December 2018 |
Institution: | Università degli Studi di Napoli Federico II |
Department: | Scienze Chimiche |
Dottorato: | Biotecnologie |
Ciclo di dottorato: | 31 |
Coordinatore del Corso di dottorato: | nome email Sannia, Giovanni sannia@unina.it |
Tutor: | nome email Tutino, Maria Luisa UNSPECIFIED |
Date: | 8 December 2018 |
Keywords: | Strain engineering; vectors development; protein production; protein purification |
Settori scientifico-disciplinari del MIUR: | Area 05 - Scienze biologiche > BIO/10 - Biochimica Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare Area 03 - Scienze chimiche > CHIM/11 - Chimica e biotecnologia delle fermentazioni |
Date Deposited: | 15 Jan 2019 09:11 |
Last Modified: | 27 Jun 2020 05:00 |
URI: | http://www.fedoa.unina.it/id/eprint/12510 |
Collection description
Neurological disorders constitute the major cause of disability adjusted-life years (DALYs). Alzheimer’s disease and other dementias are included in the group of the most prevalent disorders in this field (Feigin et al., 2017). Nevertheless, the urgency of the treatment of these pathologies is not met by efficacious drug development, as indicated by a recent statistical analysis measuring the likelihood of approval of new drugs by disease area (Hay et al., 2014). In neurology only 8.4% of the candidate drugs have been approved in the 2006 – 2015 decade and the drugs categorized as “Large Molecules” were characterized by 13.2% rate of success (Hay et al., 2014). This statistical analysis would suggest that only those molecules proving robust Proof of Mechanism (PoM) and Proof of Principle (PoP) during their early development deserve the risk for further development. On the other hand, proteins used in protein replacement therapies (PRTs) constitute an exception in this scenario as the iter for their approval is generally more straightforward (Gorzelany and De Souza, 2013). In this case the main hurdles involving the drug development directly coincide with the production, the purification and the stable formulation of the final product rather than the assessment of its efficacy or toxicity (Saccardo, Corchero and Ferrer-Miralles, 2016). In the present document we describe the approaches followed in the implementation of biotechnological processes for the production of two different proteins with potential applications in the treatment of central nervous system (CNS) disorders. In the first chapter we present the establishment of a simple and efficient pipeline for the E. coli recombinant expression and purification of the bacterial toxin named CNF1. This 114 kDa protein is involved in a series of infectious diseases (Ho et al., 2018), but it has also been demonstrated to be promising in the treatment of severe neurological pathologies like Alzheimer’s disease, Parkinson disease, Rett syndrome and epilepsy (Maroccia et al., 2018). Nevertheless, during the development of the project we decided not to pursue any further attempt in the clinical development of CNF1 as a drug because of the lack of robust, clear and completely demonstrated PoM and PoP. However, the proposed procedure for the purification and final formulation of the product outperforms the others reported in the literature in terms of yield, purity and stability and it can be easily employed in the future for further structural and functional analyses in toxicological and immunological perspectives. The reproducibility of the entire pipeline has been demonstrated repeating the production and purification protocols dozens of times at intervals of several months. Moreover, the stability of the final product was routinely ascertained using SDS-PAGE, size-exclusion chromatography, DLS and activity assays. In the second chapter of this thesis we describe the employment of Pseudoalteromonas haloplanktis TAC125 in the production of a human protein to be used in a PRT. Although the quality of the product achievable in this host seemed better than the one previously obtained with other expression systems, the overall yields remained low. Slight improvements in this sense were achieved by the genetic engineering of the coding sequence of the target protein and by the implementation of alternative expression plasmids. Nevertheless, further studies demonstrated that the whole expression platform – the host and the plasmids – are affected by imperfections and bottlenecks whose correction is pivotal for a satisfying recombinant production. This kind of issues is typical of unconventional and less explored recombinant bacteria, but there are several examples in the literature about how they can be systematically overcome. Hence, a series of measures to be taken for the improvement of this microbial factory are proposed.
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