Biology Reference
In-Depth Information
convenient for the purpose of recombinant adenovirus production; similar kits are
available from other manufacturers. As complete and detailed instructions are given
with these commercial products, including the provision of vectors for the insertion
of the sequences to be expressed and the cell lines required for recombinant viral con-
struction and propagation, no description is provided here. However, as in the case of
recombinant baculovirus, we recommend that both the titer of the amplified recombi-
nant viruses and the length of time of infection be optimized for production of recom-
binant TBCD. Even under optimal conditions, the yield of recombinant TBCD is rarely
better than 1-5% of the total protein. Nonetheless, even at the low end of this range, it
is possible to purify this material to homogeneity as described below.
11.1.7
Purification of TBCD
11.1.7.1
Preparation of HeLa cell lysates
The preparation of a particle-free lysate of adenovirus-infected HeLa cells is iden-
tical to that used for the preparation of lysates from infected insect sf9 cells. Follow
the steps described in
Section 1.5.1
a-j.
11.1.7.2
Anion exchange chromatography on Q Sepharose High
Performance
Purification of recombinant TBCD on Q Sepharose High Performance (QHP) follows
the same procedure as that described for TBCE (
Section 1.5.2
). TBCD emerges from
the QHP column in the conductivity range 17.5-21.5 mS/cm. The relatively low yield
of the recombinant protein in adenovirus-infected HeLa cells make it advisable to lo-
cate it as a 116 kDa band by Western blotting using an anti-TBCD antibody.
11.1.7.3
Anion exchange chromatography on MonoQ
a.
Pool the fractions identified as containing TBCD and concentrate to 2 ml using a
Millipore Centricon device (Millipore Inc.) or equivalent.
b.
Exchange the protein into 20 mM NaP04 buffer, pH 7.5 using a PD10 desalting
column (GE Healthcare Inc.).
c.
Apply the sample to a 0.5
5.0 cm (5/5) MonoQ column (GE Healthcare Inc.).
Wash and elute the column as described in
Section 1.3.2
c. TBCD emerges from
this column in the conductivity range 19.0-22.0 mS/cm and can be detected
either by Western blotting with an anti-TBCD antibody or as a Coomassie stained
band migrating at 116 kDa.
11.1.7.4
Chromatography on hydroxylapatite
a.
Pool the fractions identified as containing TBCD and concentrate to 2 ml using a
Millipore Centricon device (Millipore Inc.) or equivalent.
b.
Exchange the protein into NaP0
4
buffer, apply this material to a hydroxylapatite
column, wash and develop as described in
Section 1.5.3
b-d. TBCD emerges as an
identifiable absorbance peak in the conductivity range 1.5-6.5 mS/cm.
