Strategies to improve Chlamydomonas reinhardtii as a recombinant protein host: from a small growth factor to a complex monoclonal antibody production
[eng] Chlamydomonas reinhardtii has emerged as a promising alternative host for recombinant protein expression. Despite its advantageous characteristics and low-cost production, its use is hampered by low expression levels of nuclear transgenes. In this thesis we test several strategies designed to...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2018 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/123516 |
| Acceso en línea: | https://hdl.handle.net/2445/123516 http://hdl.handle.net/10803/586310 |
| Access Level: | acceso abierto |
| Palabra clave: | Proteïnes recombinants Anticossos monoclonals Factors de creixement Recombinant proteins Monoclonal antibodies Growth factors |
| Sumario: | [eng] Chlamydomonas reinhardtii has emerged as a promising alternative host for recombinant protein expression. Despite its advantageous characteristics and low-cost production, its use is hampered by low expression levels of nuclear transgenes. In this thesis we test several strategies designed to reduce or overcome this limitation. As a result, on the base of a secreted fusion protein comprising a small growth factor and a reporter, the use of regulatory and stabilizing regions resulted in expression levels ranging from 1 to 100 µg /L of culture. We report the expression of a fully-assembled monoclonal antibody in Chlamydomonas nucleus, therefore, validating Chlamydomonas as a host for complex protein production. The cassettes and high throughput screenings developed emerge as innovative tools expanding the molecular toolbox available for Chlamydomonas nucleus. In addition, a scalable purification method to recover the target protein from culture medium has been developed and validated indicating a simple downstream processing for secreted recombinant protein production. Finally, we report that Chlamydomonas secreted components induce proliferation of murine fibroblasts and have a synergistic effect with supplied hEGF, unveiling the potential of extracellular components of Chlamydomonas for a variety of applications. |
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