Expression and purification of the wild-type and mutant XynA proteins
The recombinant plasmid pET28a-XynA was transformed into BL21-Codonplus (DE3) competent cells, and then a single colony was selected and inoculated into 100 mL of LB medium containing 30 μg/mL kanamycin (Sangon Biotech, Shanghai, China) and 32 μg/mL chloramphenicol (Sangon Biotech, Shanghai, China) and cultured in a 37 °C constant temperature shaking table at 180 rpm for 10 hours. When the OD600reached 0.6-0.8, the E. coli had reached the logarithmic growth stage. Protein expression was then induced with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Sangon Biotech, Shanghai, China) at 25 °C on a constant temperature shaker for 12 hours. Finally, the samples were collected by centrifugation at 4000 rpm for 20 minutes at 4 °C and stored at -80 °C until use.
The collected E. coli was suspended in buffer A containing 40 mM Tris-HCl at pH 8.0 with 250 mM NaCl and 10 mM imidazole at 4 °C, and then 1 mM β-mercaptoethanol (Amresco, Guangzhou, China) and 1 mM PMSF (Sigma, Beijing, China) were added. The resuspended E. coli cells were passed through a low-temperature ultrahigh-pressure cell disrupter at 1200 psi, and the obtained lysate was centrifuged at 14000 rpm for 60 minutes (4 °C). After centrifugation, portions of the supernatant and the precipitate were retained (Coomassie brilliant blue: running glue samples) (Laemmli, 1970). The supernatant was divided into two parts: one part was directly combined with nickel-nitrilotriacetic acid (Ni-NTA) affinity resin (Qiagen, China); the other part was heated in a 65 °C water bath for 60 minutes and then centrifuged at 14000 rpm for 60 minutes. After the second centrifugation, a part of the supernatant and the precipitate were retained, and the supernatant was combined with Ni-NTA. The two supernatants above were combined with Ni-NTA for 45-60 minutes. The hybrid protein was removed by using buffer A (40 mM Tris-HCl at pH 8.0 with 250 mM NaCl, 10 mM imidazole at 4 °C), and the target proteins were eluted by using buffer B (40 mM Tris-HCl at pH 8.0 with 250 mM NaCl, 250 mM imidazole at 4 °C). XynA was purified in two ways, one of which was heating in a 65 °C water bath for 60 minutes and the second was using a Hi Trap QHP column (1 mL GE Healthcare, United States). The protein is bound to the column at loading. The conditions are then changed, and the bound components elute separately. Purification was carried out on a 1 mL QHP column with a NaCl gradient, and the protein solution was injected into the equilibrium column at a flow rate of 0.5 mL/ minute. A linear NaCl gradient elution of 50 to 250 mM was used, and 2 mL fractions of the eluate were collected over the entire gradient length.
The state of XynA in solution was analyzed by a Superdex 200 (10/300) increase column (GE Healthcare, United States). First, the chromatographic column was balanced with a buffer containing 150 mM NaCl, 40 mM Tris-HCl at pH 8.0 and 1 mM dithiothreitol (DTT, Sangon Biotech, Shanghai, China). Second, XynA loading. Finally, the column was eluted with buffer solution, and 2 mL fractions of eluate was collected.