Hiprep cm ff 16 10 column

Wild-type and mutant forms of Xln A were expressed and purified from the supernatant of S. lividans using a slightly modified version of a previously reported protocol (12 (link)). Genetic constructs for cysteine mutants at positions K48 and S212 were a generous gift from Prof. Claude Dupont (INRS). Briefly, S. lividans was first pre-cultured in 12 mL TSB medium for 2 days and further expressed for 72 h at 34°C by inoculation in M14 minimal medium using xylose as the sole carbon source. The culture supernatant was concentrated by ultrafiltration and dialyzed against 20 mM citric buffer (pH 4.5). The dialyzed enzyme solution was then loaded on a HiPrep CM FF 16/10 column (GE Healthcare) pre-equilibrated with 20 mM citric buffer (pH 4.5). The enzyme was eluted with a linear gradient of the same buffer containing 1 M NaCl. Fractions were collected and protein elution was monitored at 280 nm. Elution fractions were further analyzed by SDS-PAGE and fractions containing Xln A were collected, pooled, dialyzed against water, and lyophilized.