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Author
Grau García, Paula
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Abstract
Insect cells have been widely used as a platform for the production of recombinant proteins requiring post-translational modifications, basically using the well established BEVS (Baculovirus Expression Vector System), yielding high productivities. Further advantages of this system are that cells can reach high concentrations, high production yields and culture media have lower cost when compared to other expression systems such as mammalian cells. However, the preparation of viral stock and the additional purification burden associated to the need of separating the baculoviruses from the produced protein are the main drawbacks in using this platform. The motivation of the work presented here has been to explore the development of a production system using insect cells but free of baculovirus.
Stable gene expression (SGE) is the selected approach to obtain stable cell pools producing recombinant proteins. With this, a constitutive gene expression is achieved via integration of the gene of interest into the cell genome by random integration. Then, protein expression can be further improved by enriching the pool with a higher proportion of producing cells through cell sorting selection.
In the present work, the production capacity of two stable insect cell lines , Spodoptera frugiperda (Sf9) and Trichoplusia ni (Hi5), in suspension cultures to produce two different protein models, mCherry and Gag::eGFP, has been studied. These cell lines are the most common insect cells used in protein expression, and the two proteins have been selected because they have very different structure, mCherry being a simple protein while Gag::eGFP is forming a polyprotein structure, named as Virus Like Particle (VLP) processed internally by the cell and exported through the cell membrane via a budding process. On top,both proteins have a fluorescent nature, what facilitates the traceability of its production and cell sorting via flow cytometry.
The different cell populations developed in this work are compared in terms of fluorescence in order to analyse the concentration of the protein produced. The percentage of recombinant cells and mean fluorescence intensity is also analysed by flow cytometry. Since Gag:eGFP is an autoassemblable and secretable polyprotein, the budding capacity of both cell lines is also studied by measuring the fluorescence in the supernatant, and also was qualitatively studied by cryo-transmission electron microscopy (cryo-TEM).
The obtained results indicate that cell clones developed with this approach are good candidates for both simple and complex stable recombinant protein production, obtaining a total protein concentration of 2.68 · 103 ± 0.45 μg / mL in Sf9 cells and 1.63 · 103 ± 0.29 μg / mL in Hi5 in the case of mCherry protein. The concentrations obtained from Gag :: eGFP were 7.48 ± 0.27 μg / mL in Sf9 and 14.77 ± 0.34 μg / mL in Hi5. When the secretion of Gag::eGFP was studied, it was seen that in the Hi5 cell line the levels were not detectable with the techniques commonly used, and were detected at quite low levels in the case of Sf9,being 7.48 ± 0.27 μg / mL what represented a production of 1.04 · 103 ± 0.56 ng / mL of VLPs. This observation seems to indicate that these cells present some bottle-neck in the processing of complex molecules in the membrane.
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