The pRSF-1b plasmid features coexpression capabilities as well as the ability to express fusion proteins with a N-terminal His?Tag coding sequence that results in native protein after purification and cleavage. This plasmid carries an origin derived from RSF1030 (1, 2) and kanamycin resistance, allowing for the option of coexpression with many other Novagen T7-based expression vectors. Please read technical bulletin, TB401, for all possibilities and contact technical service if you need additional information.
This plasmid contains a strong T7lac promoter, an amino-terminal His?Tag coding sequence, and multiple cloning site (MCS) regions designed to allow the generation of target proteins with minimal vector encoded fusion. The Pml I cloning site allows direct fusion to the His?Tag sequence for inserts that are blunt and in the appropriate reading frame. For applications requiring a removable amino-terminal His?Tag sequence, the MCS includes a PshA I cloning site (GACNNNNGTC). The PshA I site overlaps the cleavage site for the enterokinase (EK) protease (AspAspAspAspLys). Cloning appropriately designed inserts into this site re-creates the full EK site and allows all amino-terminal vector-encoded sequences to be removed by EK digestion. The remainder of the MCS encodes restriction enzyme sites found in many other Novagen expression vectors to facilitate insert transfer. An optional S?Tag coding sequence is present at the distal end of the MCS for generating a carboxy-terminal tag compatible with purification, detection, and quantification (3).
