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.