The pET-52b(+) vector carries an N-terminal Strep•Tag® II coding sequence (1) followed by a recognition site for the human rhinovirus (HRV) 3C protease. This protease is highly specific for cleavage of the sequence LEVLFQ↓GP (2), and is active at low temperatures (3). The multiple cloning region is followed by an optional thrombin recognition site and a C-terminal His•Tag® coding sequence. The presence of two “gentle elution" tags at both the N-terminus and C-terminus is ideal for dual purification strategies designed to isolate full-length fusion proteins (4). Unique restriction sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circle map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below the circle map. The f1 origin is oriented so that infection with the helper phage will produce virions containing single-stranded DNA that corresponds to the coding strand.
