Structural Analysis and Mutagenesis of PB2SF

The protein sequence of PB2SF was uploaded, and a three-dimensional model of PB2SF was built by the protein structure homology-modeling server SWISS-MODEL online¹ (Biasini et al., 2014 (link)) using the structure of bacterial 2-O-sulfatase BT_1596 as a template (PDB code: 3b5q) (Ulmer et al., 2014 (link)). Structural figures were created and rendered using the program PyMOL (Schrödinger LLC²).
Mutants of PB2SF were produced by using the Fast Mutagenesis System Kit (TransGen) as described by Prabhakar et al. (2005) (link). The primer sequences used for the mutations are presented in Table 2. The plasmids of the mutants were amplified by transferring them to DMT Chemically Competent Cells (TransGen), and clones were sequenced at Sangon Biotech, Co., Ltd. (Shanghai, China) to confirm the successful introduction of the mutations. Then, correct plasmids were transferred into E. coli BL21 (DE3) cells and expressed, and the enzymatic activity of the mutants was determined under optimum conditions [50 mM HAc-NaAc buffer (PH 6.0), 40°C], as described above for the wild-type enzyme PB2SF.