Biology Reference
In-Depth Information
The recombinant protein should be visible as a conspicuous band migrating with an
apparent mass of 62 kDa.
11.1.5.3
Chromatography on hydroxylapatite
a.
Pool the fractions containing TBCE from the anion exchange column and
concentrate them to 2 ml using a Millipore Centriprep device or equivalent.
b.
Exchange the protein into 10 mM NaP0
4
buffer, pH 7.5, 0.1 mM MgCl
2
,1mM
DTT using a PD10 desalting column (GE Healthcare Inc.).
c.
Apply the desalted sample to an FPLC column of hydroxylapatite (Pentax,
American Chemical Co., Inc.) (0.5
2.0 cm) equilibrated in 10 mM NaP0
4
buffer, pH 7.5, 0.1 mM MgCl
2
, 1 mM DTT using the 2 ml loop.
d.
Wash the column after sample loading with 2 ml of equilibration buffer and
develop with a 20 ml linear gradient of equilibration buffer containing 0.25 M
NaP0
4
, pH 7.5. Collect fractions of 1 ml. The column may be run at room
temperature, but it is advisable to transfer the fractions to an ice bucket as soon as
they emerge from the column.
e.
TBCE emerges from the Pentax column as a symmetrical peak in the conductivity
range 2.4-4.7 mS/cm.
f.
Analyze a small proportion (e.g., 2.5
l) of the samples contained within this
conductivity range by SDS-PAGE to determine the purity and yield of the product.
m
11.1.5.4
Size exclusion chromatography (optional)
a.
Pool the fractions containing TBC from the hydroxylapatite column and
concentrate them to 0.5 ml at 4
C using a Millipore Centricon device or
equivalent.
b.
Apply this material to a Superdex 200 gel filtration column (22 ml) equilibrated
in 10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 1 mM DTT. Collect
0.5 ml fractions.
c.
TBCE emerges from the column as a symmetrical peak at 13-14 ml and should
have a purity of 98% or better as judged by SDS-PAGE. This material can be
stored flash frozen in small aliquots at
70
C.
11.1.6
Expression of TBCD
TBCD expressed in
E. coli
is insoluble and cannot be recovered by renaturation. We
have also attempted to express TBCD as a recombinant protein in insect sf9 cells.
While this results in a modest yield of recombinant protein that is soluble, we found
this material to be unstable (for a reason that is unclear) in the sense that attempts to
purify it by any chromatographic procedure resulted in massive losses. We therefore
adopted a procedure that depends on the use of recombinant adenovirus engineered for
the expression of TBCD in HeLa cells. We have expressed both human and bovine
TBCD via adenovirus vectors, and both are biologically active, although they have
distinctive properties upon expression in cultured cells (
Tian et al., 2010
). We have
found the AdEasy kit sold by Stratagene Inc. to be relatively straightforward and
