Expression Plasmid
To express our proteins we used a pET24D(+) vector and TEV-protease system to express our smaller proteins with a cleavable GFP. The pET24D(+) vector is Kanamycin resistant and also allows our protein to be expressed with a 6-residue His-Tag for affinity chromatography purposes. All of our proteins will be expressed using E.coli K-12 strain. For more information see our protocol on expression and purification.
pET24D + vector with fluorescent synechococcus phosphate binding protein (PiBP) inserted. Plasmid sequence obtained from the Addgene vector database (#69752-3) and sequence was reversed for a forward orientation of the T7 promoter start site. Fluorescent PiBP was inserted into the plasmid to be transcribed by bacterial RNA pol after the ribosomal start site (cut site NcoI). XhoI cut site inserted at the C-terminus site of fluorescent PiBP, for expression of the protein with a 6-residue His-tag. Vector contains Kanamycin resistance.
Tobacco etch virus (TEV) protease system for small protein expression. Protein structures were obtained from the RCSB Protein Data Bank. Coiled-coil image generated using PyMOL. Images from L-R: 6 residue Histidine tag, green fluorescent protein, TEV-protease (grey) with ENLYFGQ (black), coiled-coil.TEV-protease system used to express small proteins such as a 35-residue coil where otherwise bacterial expression may prove difficult. His-tag and GFP are attached to a cut site ‘ENLYFGQ’ for the TEV-protease. His-tag and GFP to be cleaved by TEV-protease after purification by nickel chromatography leaving a functional coil protein on its own.