| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2004: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
(1)Library screening
For the second step of the screening, an experimental system was established to express proteins in E coli cells a library of proteins with the following repetitive sequences :
(Gly-Gly-X1-X2-X3-Gly-X4-Pro-X5-X6-Asn-Ala)n,
where X1,IIe,Leu,Val, or Phe ; X2,Ser or Thr ; X3,IIe,Leu,Val, or Phe ; X4,Ala or Gly ; X5, Arg or His ; X6,Glu or Val ; n>10.
After screening the library, nine clones were found to express soluble proteins, and their nucleotide sequences were determined. Some of these clones produced soluble proteins with the number of repetition of>50.
(2)Purification of the soluble proteins
The His-tag sequence was added at the C termini of the proteins, and they were purified by His-bind chromatography followed by hydroxyapatite chromatography.
(3)Structural analysis of the proteins and search for possible functions
The structures of the purified proteins were analyzed by circular dichrosim spectroscopy measurements to find these proteins have random coil structures. These proteins with repetitive sequences were designed to have hydrophobic residues at regular intervals. Thus, it is possible the proteins can bind small hydrophobic molecules with repetitive structures. I am now studying if the proteins bind them and if structural changes are elicited upon binding these small molecules.
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