Cloning and characterization of Trypanosoma brucei IFT complex B core

摘要

The cilium, a protrusion from the cell surface, has recentlyattracted immense attention due to its importance in cellsignaling and its association with a wide range of humandisorders. On the structural level, the cilium consists of aring of nine microtubule doublets along which the bidirectionalmovement of ciliary building blocks and turnoverproducts takes place. This transport is termed intraflagellartransport (IFT) and is carried out by two distinctmulti-subunit protein complexes, IFT complex A and B,which are responsible for retrograde and anterogradetransport, respectively. IFT complex B consists of at least14 different proteins, eight of which are known to form asalt-stable core complex. Our research focuses on the IFTcomplex B core. The eight IFT genes from Trypanosomabrucei have been cloned into 4 Duet vectors (Novagen) toenable a simultaneous co-expression of the core complex.Upon co-expression in E. coli, the complex has been purifiedwith minor impurities using Zinc-NTA affinity andsize-exclusion chromatography. Six of the eight proteinshave been confirmed via mass spectrometry and Westernblot. On size-exclusion chromatography, different oligomericstates of the complex can be observed, ranging from>8MDa to around 600 kDa. Preliminary data suggestsa liposome-binding capability of the complex, but theresponsible subunit(s) remains to be elucidated. Sincecrystallization of the purified complex has not been successful,future studies will focus on electron microscopyanalysis of the recombinant IFT complex B core.