The pTWIN vectors are designed to generate reactants for IPL, however the performance of the IPL reaction itself will vary significantly depending on the protein or peptide reactants used. For intermolecular IPL reactions, ones that do not involve cyclization, it is necessary to have the reactants as concentrated as reasonably possible. In an ideal case, at least one of the reactants should be close to a concentration of 1 mM. Because the cyclization is an intramolecular reaction, the reactant concentration is not critical. By cloning a gene into the appropriate pTWIN restriction sites it is possible to isolate a target protein with an N-terminal cysteine (fusion with intein 1), a C-terminal thioester, or both. The presence of both an N-terminal cysteine and a C-terminal thioester allows the in vitro cyclization of a target protein with a peptide bond at the site of fusion.
Studies by NEB scientists have indicated that pTXB vectors are more suitable for IPL than the pTYB vectors because they cleave more proficiently with thiol reagents that are best for ligation, such as 2-mercaptoethanesulfonic acid (MESNA) and thiophenol. Cleavage of fusion proteins expressed from a pTYB vector may require a higher incubation temperature with these thiol reagents. However, it may be advantageous to express a protein of interest using both pTXB1,3 and pTYB1,2,3,4 vectors (cloning the insert using the same restriction sites) and examining both constructs for expression and purification.