Optimisation de la production de proteines recombinantes avec des cellules de tabac en suspension.

摘要

The continuously increasing demand for bioactive recombinant proteins at low cost stimulates the industry to develop new production platforms. Among them, in vitro plant biotechnology is gaining interest. Allowing the use of a fully synthetic protein-free medium that do not promote the development of human pathogens, plant cells can perform post-translational modifications and lead to bioactive therapeutics. Various human proteins produced in plant cell cultures are currently under clinical trials. However, low recombinant protein yields obtained with plant cells limit the competitiveness of this new platform.;In order to present an overview of the actual situation in the industry, an exhaustive literature review was produced and submitted to the Plant Biotechnology Journal (February 2010). In the first part of this review manuscript, all the established recombinant protein production platforms as well as the main emerging platforms are described in detail with their specific applications. Upcoming innovations for the in vitro plant biotechnology platform such as the optimization of expression cassettes, of bioprocess parameters and the use of viral suppressors are then presented and discussed.;In a second part, a chapter presents a study showing the feasibility of transiently producing recombinant proteins, in plant cells in suspension, by cocultivating them with Agrobacterium populations capable of transferring plasmids containing various genes of interest. It was shown that transiently expressing a viral suppressor like p19 and a gene of interest simultaneously significantly raises recombinant protein yields. The second manuscript, submitted to Biotechnology Progress (February 2010), presents a study on these two complementary approaches. Nicotiana benthamiana cells were cocultivated with two different Agrobacterium strains, one bearing a construct for the expression of a gene of interest, a murine IgG1, and the other one bearing the viral suppressor p19. Using this coculture transient system, it was shown that co-transformation of plant cell suspension culture with two transformation vectors is feasible and that the use of a viral suppressor of silencing significantly raises the recombinant protein production similarly to what is observed in agroinfiltrated plant leaves. It was also showed that using a transient approach for the expression of the viral suppressor may be preferable to the establishment of a stably expressing cell line due to the fact that the silencing suppressor effect of p19 is related to its expression level and that this level was significantly lower in the stable cell line than in the transiently transformed one.;In parallel to transient cocultures, the identification and isolation of strong and cold-inducible promoters in N. tabacum was attempted. As shown in the review manuscript, cold-induced production systems are being developed in microbial and mammalian cells; cold being identified as a parameter allowing a raise of active and correctly folded recombinant protein while reducing the work of proteases. No such system has, to our knowledge, been made for plant systems in a perspective of recombinant protein production. Plant cells are normally grown between 23-27°C and the effects of a cold stress at 12°C or 18°C for 6, 12, 24 and 48 hours on plant biomass, total extracellular protein concentration and pH were characterized. Using a Differential Display approach, fragments of the 3'-untranslated region (UTR) of a number of potential cold-inducible genes have been isolated. Validation of these candidates has been attempted using Northern or Reverse Northern blots with no results. Databases were screened using a BLAST strategy, and allowed a reduction to a few potential candidates. A 5'-Rapid Amplification of cDNA ends (5'-RACE) method was performed on these candidates in order to get a longer gene sequence. However, none of the isolated fragments using this method were related to the candidates.;The research in this thesis is aimed at raising recombinant protein yields from tobacco cells in suspensions. The main objective was to optimize recombinant protein production in tobacco cells in a bioreactor process. Secondary objectives were to study plant cell coculture with Agrobacterium to transiently express a gene of interest and a viral suppressor and to identify and isolate strong cold-inducible promoters.;In this thesis, the feasibility of a transient expression system that combines plant cells with many Agrobacterium strains was demonstrated as well as the importance to transiently express a viral suppressor in order to raise recombinant protein yields. However, much work remains to be done in the identification and isolation of cold-inducible promoters.