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Baculovirus co-expression and purification of CDK5 and p35nck5a

Authors Leung, Yuk Fai
Issue Date 1998
Summary Nclk is a heterodimer of a catalytic subunit cdk5 and a 25 kDa regulatory subunit, which is a truncated form of a 35kDa protein. A 39kDa isoform of the 35kDa protein was also found in the brain. The expression patterns of these two cdk5 activators are neuron specific, and spatially and temporally regulated during embryonic development. Recent research has shown nclk is involved in the regulation of the neuronal and muscle differentiation. Abnormal regulation of nclk has been implicated in some neurodegenerative diseases like Alzheimer's disease. Attempts in expressing these activators by bacteria for in vitro characterization were unsuccessful because most of the expressed protein was truncated. Full-length activators were expressed by baculovirus expression system in this study. Baculovirus expression system has several advantages, for example, it is an eukaryotic expression system and would have better post-translational modifications for the recombinant protein. It also allows the co-expression and reconstitution of full-length activators and cdk5 simultaneously in the cell. Singly expressed and purified cdk5, when reconstituted with bacterially expressed GST-p25, was shown to have comparable kinase activity to the authentic nclk originally purified from bovine brain. On the other hand the singly expressed activators were not stable and would truncate quickly. Besides, they could not activate bacterially expressed GST-cdk5 substantially. Nevertheless, the activators could be stably co-expressed with cdk5. The attempt to develop a protocol for purification of the expressed recombinant proteins is described. CdkYp35 can be purified to a state that possess comparable specific kinase activity to nclk purified from bovine brain by passing through a Hi-Trap SP cation exchange and Ni-NTA affinity column.
Note Thesis (M.Phil.)--Hong Kong University of Science and Technology, 1998
Language English
Format Thesis
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