Purification of MBP fusion proteins using engineered DARPin affinity matrix.
- 1. Center for Interdisciplinary Biosciences, Technology and Innovation Park of P.J. Šafárik University, Jesenná 5, 041 54 Košice, Slovakia Department of Biophysics, Faculty of Science, P.J. Šafárik University, Jesenná 5, 041 54 Košice, Slovakia
- 2. Institute of Neuroimmunology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 10 Bratislava, Slovakia
- 3. Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
- 4. Center for Interdisciplinary Biosciences, Technology and Innovation Park of P.J. Šafárik University, Jesenná 5, 041 54 Košice, Slovakia
Description
Maltose binding protein (MBP) has a long history as an expression tag for the production of recombinant fusion proteins. The ability of MBP to increase the solubility of fusion proteins makes it a preferred option for the expression of recombinant proteins that are unable to fold properly or that tend to form aggregates. A critical step for obtaining a sufficient amount of the MBP fusion protein is the purification. Commercially available affinity chromatography for the purification of MBP fusion proteins using amylose matrix has two main issues: (i) low (micromolar) affinity and (ii) limited number of uses due to the cleavage of polysaccharide matrix by the amylases present in the crude cell extract. To avoid this problem, we developed an affinity chromatography based on the protein-protein interaction. We use an evolved Designed Ankyrin Repeat Protein off7 (DARPin off7), which interacts with MBP with almost 1000 times higher affinity than amylose. The functionality of such affinity chromatography was tested with the purification of MBP-tagged green fluorescent protein (GFP) and flavodoxin (FLD). The optimized affinity chromatography based on the MBP-DARPin off7 interaction enables the purification of the fusion proteins in a single-step procedure. This affinity column - easy to construct, resistant to amylase and reproducible for numerous purification cycles - provides an alternative approach to commercially available affinity chromatographies for purification of proteins containing the MBP tag.
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MS_MBP-for Zenodo.pdf
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- Journal article: 10.1016/j.ijbiomac.2021.07.117 (DOI)