Biology Reference
In-Depth Information
CHAPTER
17
Purification and Assembly
of Bacterial Tubulin
BtubA/B and Constructs
Bearing Eukaryotic Tubulin
Sequences
Jos ´ M. Andreu and Mar´a A. Oliva
Centro de Investigaciones Biolo´gicas, CSIC, Madrid, Spain
CHAPTER OUTLINE
Introduction and Rationale....................................................................................... 270
17.1 Materials: Genes, Constructs, and Expressed Proteins...................................... 272
17.2 Methods ........................................................................................................ 273
17.2.1 BtubA/B Purification.................................................................. 273
17.2.2 BtubA and BtubB Purification .................................................... 275
17.2.3 BtubA/B Chimera Containing Eukaryotic Sequences ..................... 276
17.2.4 Methods to Study Assembly of Purified BtubA/B .......................... 276
Conclusions............................................................................................................ 280
Acknowledgments ................................................................................................... 280
References ............................................................................................................. 280
Abstract
Bacterial tubulin BtubA/B is a close structural homolog of eukaryotic
-tubulin,
thought to have originated by transfer of ancestral tubulin genes from a primitive
eukaryotic cell to a bacterium, followed by divergent evolution. BtubA and BtubB
are easily expressed homogeneous polypeptides that fold spontaneously without eu-
karyotic chaperone requirements, associate into weak BtubA/B heterodimers and as-
semble forming tubulin-like protofilaments. These protofilaments coalesce into pairs
and bundles, or form five-protofilament tubules proposed to share the architecture of
microtubules.
Bacterial tubulin is an attractive framework for tubulin engineering. Potential ap-
plications include humanizing different sections of bacterial tubulin with the aims of
ab
