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CONCLUSIONS
In this chapter, we have describedmethods for purification of bacterial tubulinBtubA/
Band chimera containing eukaryotic tubulin sequences, aswell as for characterization
of their assembly, aiming to facilitate tubulin engineering for microtubule research,
based on easily expressed bacterial constructs. In principle, any different eukaryotic
tubulin sequences can be replaced into bacterial tubulin (see Section on “Introduc-
tion”;
Fig. 17.1
), although this may come at the expense of reduced solubility. For
example, we have observed how an accumulation of several eukaryotic tubulin loops,
and in particular the S9-S10 loop from
-tubulin, results in a decrease in the yield
of soluble purified chimera (
Fig. 17.3
;
Martin-Galiano et al., 2011
).
Given their close structural similarity, it is conceivable that different sections of
bacterial tubulin may be humanized with the aims of creating recombinant binding
sites for antitumor drugs (
Jordan &Wilson, 2004
), obtaining well-defined substrates
for the tubulin posttranslational modification enzymes (
Janke & Bulinski, 2011
), or
bacterially produced microtubule-like polymers that may be employed as trails for
microtubule motor proteins (
Vale, 2003
). The latter two potential applications re-
quire replacing C-terminal segments of bacterial tubulin by eukaryotic sequences.
This appears especially feasible, because the
a
(404-451) and
(394-445) C-terminal
a
b
sequences from the
-2 vertebrate tubulin isotypes are well soluble following
expression in
E. coli
, and they form in trifluoroethanol helical segments similar to
native tubulin (
Jimenez et al., 1999
). This suggests that it may be feasible to replace
these sections of eukaryotic tubulin, including helix H12 and the C-terminal tails, or
perhaps the whole C-terminal domain, into bacterial tubulin.
-1 and
a
b
Acknowledgments
We thank our collaborators A.J. Martin-Galiano and the laboratories of J. L
¨
we and J.M.
Valpuesta. Work in our laboratory was supported by Grant BFU 2011-23416 and CAM
S2010/2353 (J. M. A.) and a Ramon y Cajal contract (M. A. O.).
References
