Can XmaI be used for cloning the gene of interest into pTYB2 or pTYB4 vector?
It is not recommended to use XmaI for cloning a gene or ORF into a pTYB N-terminal fusion vector. The recognition sequence (CCCGGG) of SmaI and XmaI in the polylinker of pTYB2 or pTYB4 vector encodes for Pro-Gly adjacent to the intein sequence. When XmaI site is used for cloning, ligation of XmaI-digested insert and a XmaI-digested pTYB vector results in the regeneration of the XmaI site (forming an ORF-CCCGGG/intein-CBD fusion). Thus, the purified protein contains Pro-Gly at the carboxyl terminus. Instead, SmaI should be used to prepare the pTYB vector so that the purified protein product contains only a single extra amino acid residue, glycine. SmaI digestion of a pTYB vector leaves a blunt-end (5´ GGG/intein…) for ligation with the blunt 3´ end of the insert (forming an ORF- GGG/intein-CBD fusion). Thus, it is not correct to design a XmaI/SmaI site in the reverse PCR primer for cloning unless proline is the native carboxyl amino acid residue.
