FAQ: If my target protein is sensitive to DTT , which vector(s) should I use?

You may use the pTWIN1 or pTWIN2 vector. Fusion of an intein tag (Intein 1) to the N-terminus of a target protein allows one-column protein purification with a pH and temperature shift. No thiol reagents are required for cleavage.

The N-terminus of a target protein can be fused to the C-terminus (an Asn) of Intein1 of the pTWIN vectors (the Ssp DnaB intein). A CBD, present at the N-terminus of the Ssp DnaB intein, facilitates purification using a chitin resin. The N-terminal cysteine (Cys1) of the intein has been changed to an alanine to block the splicing reaction. The Ssp DnaB intein with this mutation undergoes a temperature and pH dependent cleavage of the peptide bond between the C-terminus of the intein and the downstream amino acid. This occurs by the cyclization of the C-terminal Asn side chain to form a succinimide ring with the concomitant breakage of the peptide bond.