Site-directed mutagenesis
The GenBank of XynA is QCO69162. The target protein was preceded by a
6xHis tag. We designed primer pairs with mutation points. The primers of
E182A were used to cause the plasmid pET28a-XynA to undergo PCR. Then,
the PCR product was digested with Dpn I
(Thermo
Scientific, Waltham, United States) for 1.5 hours at 37 °C, and the
product was heated at 80 °C for 5 minutes to denature. Finally, the
product was transformed into competent DH5α cells.
The successfully sequenced mutants
were transformed into BL21-CodonPlus (DE3) cells to express the protein.
The mutant E280A needed to be mutated based on pET28a-E182A. XynA
E182Q/E280Q had the same construction method as XynA E182A/E280A. The
purification methods of various mutants and truncations of XynA are the
same as XynA.