Protein Purification
Purification of Cel9B and its truncation mutants was explained somewhere
else (Kostylev et al., 2014). Wild type ( WT) and mutants of Cel9B
were expressed in E. coli BL21-CodonPlus (DE3)-RIPL cells
(Agilent Technologies) as a starter culture in Lysogeny Broth (LB)
medium overnight at 37 °C. This starter culture was diluted 33 times in
fresh LB, and the cells were grown at 37 °C until an OD600 ∼0.8 was
reached (approximately 3 h). At this point, isopropyl
β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of
0.8 mM, and the culture was incubated at 37 0C with
shaking for an extra 16 h. The cells were harvested by centrifugation
and resuspended in 10 mL of 20 mM sodium phosphate buffer ( pH 8.0),
0.01 M imidazole (Sigma-Aldrich), and 0.5 M sodium chloride (solution
A). The cells were lysed by sonication then placed in a 50 °C water bath
for 30 min to precipitate the E. coli proteins. The precipitate
was removed by centrifugation.
The supernatant was loaded onto a nickel Sepharose 6 Fast Flow column
(GE Healthcare) and the protein was eluted by a gradient of 0.01−0.5 M
imidazole in 50 mM NaH2PO4 buffer,(pH
8.0) and 0.3 M NaCl. Elution fractions which contain WT Cel9B and its
truncation mutants, as determined by gel electrophoresis, were loaded on
a Q Sepharose (Sigma-Aldrich) column equilibrated with 0.1 M sodium
chloride in 5 mM Bis- Tris buffer (pH 5.8), and 10% glycerol (v/v). The
protein was eluted from a 0.1−0.7 M sodium chloride gradient in 5 mM
Bis-Tris buffer (pH 5.8), and 10% glycerol (v/v). Fractions were
measured by gel electrophoresis and those that contained the WT Cel9B or
its truncation mutants were combined and concentrated using Millipore
centrifugal filter units with a 30 kDa cutoff membrane. Buffer exchange
was carried out in the same filter units by washing the concentrated
protein three times with 5 mM sodium acetate buffer (pH 5.5), and 10%
glycerol (v/v). The protein was passed through a 0.22 μm filter using a
syringe and stored at -20 °C. All enzyme concentrations were determined
by spectroscopy using a NanoDrop 1000 spectrophotometer.