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In-Depth Information
et al., 2005
)asin
-tubulin. BtubA and BtubB have indeed mosaic sequences with
intertwining features from both
a
-tubulins (
Martin-Galiano et al., 2011
). An
interesting difference between bacterial and eukaryotic tubulin, considering the
structural similarity, is that BtubA/B is expressed soluble in
Escherichia coli
and can fold and form a weak dimer
in vitro
without chaperone requirements
(
Schlieper et al., 2005
). In fact, the more divergent zones of bacterial tubulin corre-
spond to the tubulin loops involved in binding to eukaryotic cytosolic chaperonin and
in microtubule assembly (
Martin-Galiano et al., 2011
). Digging deeper into bacterial
tubulin biochemical properties has showed that BtubA/B has more primitive
assembly features, including a wider range of buffer conditions for polymerization.
The distinct loop sequences and primitive assembly properties of bacterial tubulin
support its origin from a spontaneously folding
- and
a
b
-tubulin ancestor shortly
after heterodimer duplication, possibly by horizontal gene transfer from a primitive
eukaryotic cell, followed by divergent heteropolymer evolution (
Martin-Galiano
et al., 2011
).
Purified bacterial tubulin protofilaments further associate apparently forming
filament pairs and bundles, which were observed in negative stain electron micros-
copy (
Martin-Galiano et al., 2011; Schlieper et al., 2005; Sontag et al., 2005
). How-
ever, using cryo-electron tomography, bacterial tubulin has been more recently
foundtoformfive-protofilament tubules in
Prosthecobacter
and in
E. coli
cells
and BtubA/B purified with C-terminal His tags can also form five-protofilament
tubules. Therefore, these structures have been suggested to be a primitive tubular
architecture that later evolved into the typical 13-protofilaments eukaryotic micro-
tubules (
Pilhofer, Ladinsky, McDowall, Petroni, & Jensen, 2011
). This opens the
possibility of using recombinant bacterial tubules for microtubule research,
although it would be necessary to quantify first the abundance of tubules and other
polymers. The tubule formation should be confirmed as well with untagged bacte-
rial tubulin, because the His tags interfere with BtubA/B assembly (
Schlieper et al.,
2005; Sontag et al., 2009
).
The purpose of this chapter is to describe the necessary methods for purifying
untagged bacterial tubulin and characterizing its assembly, with examples of
BtubA/B constructs bearing sequences from eukaryotic tubulin studied in the
authors' laboratory.
- and
a
b
17.1
MATERIALS: GENES, CONSTRUCTS, AND EXPRESSED
PROTEINS
The
btubA
and
btubB
genes from
P. dejongeii
DSM 1225 were cloned for biscistro-
nic expression in a pHis17 vector, leaving the intergenic region intact and not adding
any extra nucleotides to the protein genes (
Schlieper et al., 2005
). From this con-
struct, untagged BtubA and BtubB are simultaneously expressed in
E. coli
C41
(DE3) cells, induced in an exponentially growing culture at OD 0.4 (600 nm), with
1 mM isopropyl
-
D
-thiogalactoside for 3 h at 37
C. Bacterial cell pellets are
b
