Protein purification and preliminary crystallographic analysis of human Lyn tyrosine kinase.
Lyn is a member of the Src family of non-receptor protein kinase. As well as all members of the Src family, Lyn is thought to participate in signal transduction from cell surface receptors. The crystal structure of Lyn would have a better understanding of Lyn function in various cells. For the purpose of crystallization, C-terminal catalytic segment of human Lyn kinase conjugating hexahistidine purification tag (His-tag) was expressed in Sf21 insect cells. After first step purification utilizing His-tag, an anion-exchange chromatogram yielded four major peaks which had distinguishable phosphorylation manner as judged by Western blot analysis, Native-PAGE analysis and kinase activity measurements. The fractioned samples were separately examined for crystallization screening using a commercial available screening kit. The mono-phosphorylated protein was crystallized with a small rod-shaped and needle clusters. The higher phosphorylated samples corresponding to the other three fractions on the anion-exchange chromatogram were aggregated or precipitated under the above conditions. A crystal of the mono-phosphorylated sample was diffracted to 3.2A with synchrotron source at Photon Factory and a complete X-ray diffraction data set was collected. The coarse structure was solved by a molecular replacement method and further structural refinement is currently underway.
Kinoshita
Takayoshi
T
Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Gakuencho 1-1, Sakai, Osaka 599-8531, Japan. kinotk@b.s.osakafu-u.ac.jp
Miyano
Nao
N
Nakai
Ryoko
R
Yokota
Koichi
K
Ishiguro
Hiroshi
H
Tada
Toshiji
T
D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D019061:src-Family Kinases / Q000737:chemistry* / Q000302:isolation & purification*
D011994:Recombinant Proteins; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases