Laboratory of Protein Synthesis and Expression
Institute for Protein Research
Professor

Junichi TAKAGItakagi

Associate Professor

Takao ARIMORI

arimori

URL: http://www.protein.osaka-u.ac.jp/rcsfp/synthesis/
Cells receive external stimuli andprocess the information inside thecell to determine how to respond to
the external environment. In thestudy of signal transduction, themost important issue is tounderstand the mechanism by which
receptors receive information at
the cell surface (i.e., outside thecell) and transmit it to the inside of
the cell across the cell membrane.
To address this question, the groupaims to elucidate the function of the"input terminal" part of signaltransduction through structural
biology approaches using X-ray
crystallography and electron
microscopy imaging. In particular,
the group will conduct "structurefrom-function" research on proteinssuch as receptors and synaptic
components working in the brain
and nervous system, molecules
involved in neuronal cell death andaxonal guidance, and signaling
molecules involved in development
and morphogenesis of organisms.
Structure Determination of
Receptor-Ligand Complexes
The structure of the complex between theextracellular domain of a receptor and its
ligand protein contains important informationthat will lead to not only the elucidation of
the signal transduction mechanism but also
the development of drugs such as inhibitors.
In order to elucidate the interaction sites andtheir roles in binding, the structure of suchcomplexes will be determined (1) at highresolution using X-ray crystallography or (2)at low resolution using electron microscopy(EM) imaging, but in multiple conformations
simultaneously.
i) Signaling system of neuroguidance factors
and their receptors We are searching forcandidate inhibitors of semaphorins, which
are neuroaxonal guidance factors, and theirreceptor, plexin, by analyzing the complex
structure and clarifying the structural biologyof their mechanisms of action (Fig. 1).

ii) Wnt proteins are essential growth
factors for stem cell proliferation, and theirlipid modifications have made themdifficult to purify and analyze. We havedetermined the three-dimensional structureof the mammalian Wnt protein for the firsttime in the world, and are elucidating thesignaling mechanism based on thisstructure (Fig. 2).
Establishment of high-qualityrecombinant protein production
systemFigure 1: Crystal structure of the semaphorin-plexin complex (left)
and Cell morphology before and after semaphorin stimulation(middle), binding sites of pleckin B1 and its inhibitor peptide (right)

Figure 2: Predicted structure of the signaling complex (right),
combining the crystal structure of Wnt3a (left) and the cryo-EMstructure of LRP6 (center).

Extracellular proteins often require theaddition of sugar chains and disulfide bonds
to maintain their structure, making it difficult
to use simple expression systems in E. coli.
The "production" of these difficult
recombinant proteins for structural analysis
and precise biochemical and physicochemical
experiments can be achieved by the following
methods.
Figure 3: Development of the ultra-high affinity affinity purification
This will be established through (1)system "PA Tag
upgrading of animal cell culture systems, (2)development of a new affinity tag system, and(3) improvement and development ofexpression methods (Fig. 3).
Protein engineering based on
structural information
Structural information is not only useful for
elucidating the mechanism of protein function,
but is also powerful for modifying or creating
Figure 4: Structure of the new small antibody format "Fv-clasp
new functions. We are conducting research togive desired functions to proteins and to
Institute for Protein Research, Osaka University
create useful molecules that do not exist in
3-2 Yamadaoka, Suita, Osaka 565-0871, Japannature (Fig. 4).
TEL: +81-6-6879-8607
FAX : +81-6-6879-8609
Protein research is a
traditional craft!

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