Sdudy of TbPF20 in prokaryotic expression and protein-protein interaction in mammalian epithelial cell

Autor: Yu-ChengLin, 林祐丞
Rok vydání: 2013
Druh dokumentu: 學位論文 ; thesis
Popis: 101
PF20 (Paralyzed flagella protein) is an essential and conserved gene of organism with motile cilium/flagellum. For protozoan Trypanosoma brucei, a parasite living in mammalian bloodsteam, TbPF20 is essential for cytokinesis in addition to motility. Within 589 residues, bioinformatic method indicated TbPF20 containing SMC (structure maintenance of chromosome) domain and WD-repeats domain. Previous studies showed that truncated TbPF20 bearing SMC and WD-repeats domains confered cytotoxicity in mammalian epithelial cells due to G2/M arrest and centrosome disjunction failure. Furthermore, SMC domain deleted TbPF20 (SacII) construct and construct bearing WD repeat domain only (WDN) both not only conferred cytotoxicity due to G2/M arrest and centrosome disjunction failure but also reduced cell motility. These cells also showed abnormal microtubule patterns. Interestingly, SacII cells displayed delicate puncta, thin thread-like beads along cell edges. To delineate contribution of each domain on the cytotoxicity, and to confirm the discovery of image data, various deletion mutants of TbPF20 were tagged with GST protein, expressed in prokaryotic expression system. Two constructs, SMC deleted fragment (GST-Sac) and WD-repeats only fragment (GST-WD), were harvested as inclusion bodies and renatured back with urea. Through GST affinity method these TbPF20 segments as baits were used to fish proteins from MDCK lysate. The results reported that GST-Sac, GST-WD and pre-WD SMC containing fragment (GST-272) would bind some proteins in MDCK cells. Western blot revealed that katanin p60, a microtubule-severing enzyme, could be pulled down only through SacII bait confirming the image data that SacII colocalized with katanin p60. Moreover, GST-Sac, GST-WD and GST-272 also specificly bound γ-tubulin and β-actin, but not bound β-tubulin. The possibility of cytotoxicity of TbPF20 in blocking cell mitosis may due to interaction with cytoskeleton related proteins and centrosome related proteins. The detail mechanisms and other possible interacted proteins of TbPF20 await further investigation.
Databáze: Networked Digital Library of Theses & Dissertations
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