Figure 1. Schematic representation of the expression vector pMJB08, and the workflow for the expression in X. laevis oocytes and purification of mammalian transport proteins.(A) Three tags, i.e. 10x-His, FLAG and HA, and one proteolytic site for the HRV3C protease were translated in-frame at the N-terminus of our transport proteins. The HRV3C protease cleaves between the amino acids QG (in red). The vector is setup in a modular manner: i.) the FLAG tag (yellow) can be swapped by a different tag X (e.g. c-myc, HA, etc.) using the unique restriction sites SalI and NotI; ii.) the HRV3C protease cleavage site (green) can be replaced by a different proteolytic site Y (e.g. for TEV protease, thrombin, etc.) using the unique restriction sites NotI and NcoI; iii.) the HA tag (red) can be exchanged by a different tag Z (e.g. c-myc, FLAG, etc.) using the unique restriction sites NcoI and all enzymes in the multi cloning site (MCS) except XmaI; iv.) a XmaI digestion removes all tags and epitopes to obtain the original Pol1 vector. (B) Schematic representation of the workflow for the expression and purification of transport proteins in X. laevis oocytes. See Methods section for a detailed description of the different steps.
Image published in: Bergeron MJ et al. (2011)
Bergeron et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license
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