Development of an insect cell factory for the production of complex biopharmaceuticals using a synthetic biology approach
Year: 2011
Type: dissertation
Abstract: Insect cells, in particular the Spodoptera frugiperda Sf9 cell line, are a popular system for the production of biologically active recombinant proteins. However, the current technology uses baculovirus infection which has two main disadvantages: firstly, the recombinant gene is only expressed transiently during the infection cycle, after which cells die; secondly, due to the lytic nature of this system, the cellular protein processing machinery is severely compromised at the end of the infection cycle, affecting the correct formation of recombinant proteins whose expression is usually controlled by very-late baculovirus promoters. Stably transformed insect cell lines represent an alternative system for continuous protein production. However, their establishment is laborious, requiring the identification of cell clones that display the right expression properties due to random integration of the gene-of-interest. Furthermore, independently of the expression system, multimeric biopharmaceuticals continue to have very low yields, mainly because the stoichiometry between components is not properly reached in the producer cells. To overcome these issues, this report shows work towards the development of a novel Sf9 cell factory combining (i) recombinase mediated cassette exchange (RMCE) for targeted gene integration and (ii) a tetracycline (Tet) inducible system for the modulation of gene expression levels. We started by evaluating the strength of different promoters to drive GFP expression in Sf9 cells as well as different transfection protocols. The baculovirus promoter OpIE2 allowed the strongest expression when compared to others, and the efficiency of transfection was significantly higher using the lipotransfection method. From the different transfection protocols, 4 cell populations with distinct fluorescence distributions were obtained after 3 weeks under hygromycin selection. These populations were independently subject to limiting dilution and isolated cell clones were analyzed by southern blot to screen for single-copy integration of the tagging GFP construct. While the majority of clones were single copy, cassette exchange was performed in a few clones by co-transfection with flippase and the target plasmid containing dsRed. Cells were selected in the presence of neomycin and cassette exchange was confirmed by PCR of genomic DNA, revealing the absence of tagging cassette and the presence of the target cassette in the some locus. In parallel, the functionality of a tetracycline inducible system in Sf9 cells was evaluated by substituting mammalian promoters with insect cell specific promoters. However, transactivation was suboptiomal, requiring additional changes to the original system. In summary, this work reports for the first time the implementation of a RMCE system in Sf9 cells and a preliminary assessment of Tet transactivation in these cells. This new cell line will be a major breakthrough since it will combine the advantages inherent to Sf9 cell growth, continuous protein production, the ability of re-using a well characterized locus for targeted DNA integration and inducible gene expression.
Author: João Vidigal
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2275409965