| Summary: | 碩士 === 國立清華大學 === 生命科學系 === 85 === The head domains of cytoskeletal intermediate filaments (IFs)
are considered to play an important role in IF assembly and
network formation. Previously, it has been shown that the head
domain of vimentin is the major target site of phosphorylation,
and the degree of phosphorylation is increased in cells
experiencing heat shock. In order to investigate the
conformation of the head domain of vimentin and obtain firmly
evidence of the temperature-related conformation change, the
head domain has been cloned and heterologously expressed in E.
coli by pET32/BL21(DE3) expression system. The resulting
protein contains a thioredoxin fusion which can increase the
solubility of the target protein and has a calculated molecular
weight 33,279 dalton. The cloned protein has been purified by
His-Bond affinity column and identified by Western blotting.
Protein degradation during the purification process made the
desired pure head domain difficult to achieve. The conformation
of the fusion protein containing thioredoxin with head domain
vimentin, instead of the pure head domain, was analyzed by
circular dichorism. However, the expected temperature-related
conformation change was not observed clearly in the CD spectrum
of the fusion protein. The problem of proteolysis should still
be resolved to achieve the exact conformation of the head
domain.
|
|---|
