Abstract
Here, we present the full-length protocol for purifying the recombinant MOS1 transposase from insect cells used in our recent publication (Pflieger et al., 2014), which involved a N-terminal MBP-tag and maltose-affinity chromatography. Due to their overall basic properties, transposases are often difficult to purify, especially because they tend to aggregate. Since the 90s, we chose a method of purification without a denaturation step. Our first priority was to preserve the 3D structure of the protein in order to maintain its biochemical activities with the highest specific activity. Nevertheless, our production/purification made from bacteria regularly contain truncated products (or degradation products) and their levels increase with concentration of purified transposase. In contrast, production/purification made from eukaryotic cells do not contain such degradation product. We thus developed a protocol involving the pVL1392 baculovirus transfer vector and the BaculoGoldTM baculovirus expression system, allowing the expression of recombinant MOS1 from baculovirus-infected Sf21 cells.
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Acknowledgments
Dussassois-Montagne was a doctoral fellow of the French government (MRT). This work was supported by the Région Centre (InhDDE project), the French ANR (Elegineer project), the University of Tours, and the Centre National de la Recherche Scientifique.
References
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