Preparation of the Akt Kinase Fusion Protein

• Main Authors: Yu-Chih Liu, 劉昱志
• Other Authors: Hsien-Bin Huang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/62769377903313199696

碩士 === 國立中正大學 === 生命科學系暨分子生物研究所暨生物醫學研究所 === 98 === Phostensin is a protein phosphatase 1 F-actin cytoskeleton targeting subunit. Phostensin is a F-actin-binding protein that binds to the pointed ends of actin filament and modulates actin dynamics. In our preliminary results, we found that Akt can phosphorylate phostensin. In order to study the effect on biochemical activity of phostensin by Akt phosphorylation, we have to prepare the active Akt kinase. Activation of Akt is occurred at the plasma membrane where Akt is phosphorylated at Thr-308 and Ser473. However, E. coli lacks the kinases that phosphorylate Akt at Thr-308 and Ser473. Therefore, in the studies, we have over-expressed (His)6-Ub-Akt1 (T308D, S473D) fusion protein. This recombinant (His)6-Ub-Akt1 (T308D, S473D) was purified by a series of methods, including Ni2+-Sepharose, Mono-Q ionic exchange and gel filtration chromatography. The homogeneity of (His)6-Ub-Akt1 (T308D, S473D) is more than 85 % after three steps of purification. However, The kinase activity assay indicated that the recombinant (His)6-Ub-Akt1 (T308D, S473D) fusion protein cannot phosphorylate phostensin.