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THOMAS WU |
Expression, Purification, and Crystallization of the N-terminus Half of Yeast Topoisomerase II |
Topo II is a polypeptide 1429 amino acids long. It
is active as a dimer. Topo II is composed of three domains. The N-terminus
(1-410) functions as the ATPase. The central domain (410-1202) is involved
in DNA binding and contains the catalytic site. The C-terminus (1202-1429)
is involved in the transport of the enzyme through membranes and possibly
interacts with other proteins. The structure of the central domain had
been solved in 1996 by X-ray crystallography by James Bergen in the Wang
lab. The extended N-terminus domain (1-660) has been cloned and expressed
in the lab. |
The goal of the research this summer was to express,
purify, and crystallize this N-terminus domain. The domain was cloned with
a hexahistidine tag on the C-terminus of the construction. Purification
was done using nickel column and one classical column purification Crystallization
was done using sparse matrix buffers by the hanging drop and vapor diffusion
technique. |
At the end of the summer, crystallization was attempted using a number
of solutions. None was found suitable. More protein was needed, but there
was a problem of low yields. The post-doc I was working with decided to
continue the research by trying a different method of expression in the hope
of better yields. |
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