Honors College Thesis
 

Developing a methodology for expression of full-length dynein intermediate chain in baculovirus

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https://ir.library.oregonstate.edu/concern/honors_college_theses/fn1076433

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  • Cytoplasmic dynein is a 1.6MDa motor protein complex that functions in transporting cargo along the cytoskeleton. It is made up of different subunits, heavy chain (HC), intermediate chain (IC), and light chains (LC7, LC8, Tctex). The intermediate and light chains interact with accessory proteins NudE and Dynactin (through the large subunit of dynactin, P150glued) to allow for cargo attachment. Previous studies in the Barbar lab have used small sections of the Dynein-IC to show IC folds in on itself to prevent NudE and P150glued binding, and that this autoinhibition is relieved by the binding of the light chains to IC. However, limitations in traditional expression systems prevented the production of full-length IC to study binding interactions. This study aimed to overcome this problem by developing a protocol to grow full length IC using the baculovirus expression system in insect Sf9 cells. Various incubation times were studied to generate the best protocol to maximize protein yield and, for the first time, consistently produce different versions of full length IC (CT-IC-FL, IC2C, Pac11 and IC1A). 5 days was found to be the best incubation time to produce P1 virus, and 4 days was found to be ideal in generating P2 and P3 virus. Baffled flasks were found to be unnecessary and decrease growth. Binding interactions of the newly generated full-length intermediate chain with light chains, NudE, and P150glued were then studied using sedimentation-velocity analytical ultracentrifugation (AUC) to confirm the presence of autoinhibition in full-length IC. The FL AUC data also confirms the binding of light chains relieves autoinhibition, allowing accessory proteins to bind.
  • Keywords: Key Words: Baculovirus, expression, Cytoplasmic dynein, dynein intermediate chain, full length IC, autoinhibition, AUC
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