Biology Reference
In-Depth Information
11.1.2.2
Preparation of bacterial lysates
a.
Resuspend the washed bacterial pellet in 10 ml of T
10
E
1
containing a cocktail of
protease inhibitors and transfer to a French Pressure Cell. Lyse the bacteria
under 1500 psi with minimally two passes or until the lysate has no detectable
viscosity when discharged drop-wise from a Pasteur pipet.
b.
Centrifuge the lysate at 30,000
g
for 15 min at 4
C. Decant the supernatant and
g
for 15 min at 4
C in an ultracentrifuge.
c.
The particle-free supernatant can either be applied directly to the first
chromatographic dimension as described below or stored by flash freezing. The
latter is most conveniently accomplished by adding the cleared supernatant
in a drop-wise stream from a pipet directly into a small Dewar flask containing
liquid nitrogen. The resulting frozen pellets can be stored indefinitely in a
centrifuge at 200,000
70
C freezer.
11.1.3
Purification of TBCA, TBCB, and TBCC
11.1.3.1
Chromatography on either Sepharose Q (TBCA and TBCB)
or Sepharose S High-Performance (TBCC) ion exchange resins
a.
Thaw the particle-free extracts prepared as described in
Section 1.2.2
cina37
C
water bath, taking care not to let the temperature of the melt rise above 4
C. Filter
the extract through a 0.4
membrane to ensure removal of all particulate
m
material.
b.
Measure the total protein using, for example, the Bio-Rad Protein Assay reagent
(Bio-Rad Inc.). The purification procedures described below are for yields of less
than 200 mg of total protein in the starting material. Larger yields require a
prorated increase in the size of the ion exchange columns used.
c.
Apply the filtrate to a 10/10 (10 mm
10 cm) column of Sepharose Q High-
Performance anion exchange (QHP) resin equilibrated in 20 mM Tris-HCl, pH
7.2, 0.5 mM MgCl
2
, 1 mM EDTA (in the case of TBCA and TBCB) or an
equivalent column of Sepharose S High-Performance cation exchange resin
equilibrated in 10 mM NaPO
4
, pH 6.8, 1 mM EDTA (in the case of TBCC). In
either case, this can be done using either the 10 ml or 50 ml FPLC superloop.
Wash the column with at least 2 column volumes of equilibration buffer or until
the absorbance at 280 nm declines to a value of 0.2 or less.
d.
Develop the column with a 100-ml linear gradient of equilibration buffer
containing 0.5 MMgCl
2
(in the case of TBCA and TBCB) or equilibration buffer
containing 0.25 M NaPO
4
, pH 6.8 (in the case of TBCC), collecting fractions of
2 ml. The columns can be run at room temperature, but the fractions should be
stored on ice as soon as they emerge.
e.
Assay an aliquot (2-5
l) of the fractions emerging from the ion exchange
column by SDS-PAGE (12%). The approximate expected conductivity ranges for
the elution positions of TBCA, TBCB, and TBCC are 7.8-9.0, 10.4-12.8, and
9.3-10.0 mS/cm, respectively. The recombinant proteins should be readily
m
