Recombinant tubulin:
Development of the recombinant tubulin technology allows dissection of the molecular mechanism underlying the neuronal disease.
(1) Expression and purification of recombinant human tubulin
Due to the technical difficulty in generating the recombinant tubulin, the application of molecular biological methods to functional study of tubulin/microtubules has been denied for a long time. Expression and purification of isotypically pure, recombinant mammalian tubulin has been a primary challenge in the field of microtubule, drug discovery and broad area of medical science. In 2013, we succeeded to express and purify an isotypically pure human recombinant tubulin using a baculobirus-insect cell expression system. Our success paves a way to future investigation of neuronal diseases caused by mutations in tubulin genes.
We developed a method to overexpress and purify recombinant human tubulin using a baculovirus-insect cell expression system. By using dual tag system, the new method allowed us to purify 5 mg of recombinant human alpha1beta3 tubulin (99% purity) per litre of insect cell culture.
The purified tubulin is fully functional in that it can be polymerized into microtubules (Fig. C) and move on a kinesin-coated glass surface (Fig. D). This method has broad utility for functional and structural studies of mammalian tubulin. This technical achievement is beginning to bring revolution in biomedical research on microtubules and their disease-related functions.
Overexpression, purification, and functional analysis of recombinant human tubulin dimer.
Itsushi Minoura, You Hachikubo, Yoshihiko Yamakita, Hiroko Takazaki, Rie Ayukawa, Seiichi Uchimura, and Etsuko Muto.
FEBS Letters 587 (2013) 3450-3455.
(2) Investigation into the basis of human neuronal disease using recombinant human tubulin
Microtubules are essential in neuronal morphogenesis and function. The mutation in tubulin genes cause a wide range of peripheral neuropathy and brain malformation, termed "tubulinopathy". The availability of recombinant tubulin opens a way to verify the linkage between a mutation in specific tubulin gene and the disease phenotype at clinical and histological level.
In the pioneering work shown below, we succeeded to establish the critical role of the R262 residue in tubulin isotype TUBB3 for mediating kinesin interaction, which in turn is required for normal axonal growth and brain development.
Overexpression of wild type tubulin (control)
Overexpression of disease associated mutant R262A tubulin
Overexpression of R262A tubulin and suppressor mutant kinesin
Mutation in human beta3-tubulin (TUBB3) cause an ocular motility disorder termed congenital fibrosis of the extraocular muscles type 3 (CFEOM3). In patients of this disease, the oculomotor nervous system develops abnormally due to impaired axon guidance and maintenance; however the underlying mechanism linking TUBB3 mutation to axonal growth defects remains unclear.
Here, we investigate microtubule-based motility in vitro using microtubules formed with recombinant TUBB3. We find that the disease-associated TUBB3 mutation R262H and R262A impair the motility and ATPase activity of the kinesin motor. Engineering a mutation in the L12 loop of kinesin surprisingly restores a normal level of motility and ATPase activity on microtubules carrying the R262A mutation. Moreover, in a CFEOM3 mouse model expressing the same mutation, overexpressing the suppressor mutant kinesin restores axonal growth in vivo (the Figure above).
These findings establish the critical role of the TUBB3-R262 residue for mediating kinesin interaction, which in turn is required for normal axonal growth and brain development.