Biology Reference
In-Depth Information
resuspended to 15-20 mL of 50 mM Tris/HCl, pH 8.0, aliquots checked for BtubA
and BtubB expression by SDS-PAGE and stored frozen at
75
C. The plasmid car-
rying the
btubA
and
btubB
genes was employed as template to generate chimeras
containing sequences from the bovine
-2 tubulin isotypes, using internal
oligonucleotides carrying the desired mutations or insertions by the QuikChange
site-directed mutagenesis kit (Stratagene). Several BtubA/B chimeras were con-
structed in which the more divergent parts of bacterial loops were replaced by the
corresponding residues from the eukaryotic H1-S1 loop (loop 1), the S7-H9 loop
(M loop), and the S9-S10 loop (loop S) (
Martin-Galiano et al., 2011
). These sections
from
-1 and
a
b
-tubulin were, respectively, exchanged into BtubA, BtubB, or both, and
all constructions were checked by sequencing both
btubA
and
btubB
genes. These
BtubA/B chimeras were expressed as the wild-type protein, but some of them were
partially soluble and required reducing the induction temperature to 30 or 25
C.
Standard centrifugation, chromatographic and electrophoresis equipment, and a
spectrophotometer are employed for proteins purification and quantification. A fluo-
rometer, a table-top ultracentrifuge, and access to electron microscopy are addition-
ally required for the characterization of protein assemblies.
- and
a
b
17.2
METHODS
17.2.1
BtubA/B purification
Bacterial tubulin BtubA/B (without affinity tags) was initially purified by anion
exchange chromatography and a size exclusion gel filtration (
Schlieper et al.,
2005
). Later, we added a polymerization-depolymerization cycle (
Martin-Galiano
et al., 2011
), inspired by classical tubulin preparation procedures (
Miller &
Wilson, 2010
). This additional step significantly improves the protein quality by re-
moving the assembly incompetent BtubA and BtubB monomers. The following
method starts from a 2-L cell culture pellet.
1. Cells Breaking
: Once the bacterial cells pellet is completely thawed on ice, add
2 mg/mL lysozyme (Sigma) and 10
g/mL DNAse (Roche) and keep the
suspension on ice for five more minutes. Then break the cells by two passes in
a cold French press and remove the cell debris by centrifugation of the lysate
for 1 h at 100,000
m
g
,4
C.
2. Anion Exchange Chromatography
: The supernatant should be filtered through a
0.22-
m filter to avoid clogging the chromatography column, and taken to 50 mL
with Lysis buffer (50 mM Tris/HCl, pH 8) to facilitate protein binding to the
anion exchange column. This is loaded on a 10-mL home-packed refrigerated Q
Sepharose HP column preequilibrated with buffer A (20 mM Tris/HCl, 1 mM
sodium azide, pH 8.5). BtubA and BtubB are eluted around 150 mM of NaCl
concentration in a 0-50% gradient of buffer B (A plus 1 M NaCl) in 20 column
volumes at 4
C (flow, 5 ml/min) and collected in 10 mL fractions. All fractions
containing BtubA or BtubB (easily distinguished in 12% acrylamide
m
