Recombinant Reconstitution and Purification of the IFT-B Core Complex from Chlamydomonas reinhardtii.

Abstract

Eukaryotic cilia and flagella are assembled and maintained by intraflagellar transport (IFT), the bidirectional transport of proteins between the ciliary base and tip. IFT is mediated by the multi-subunit IFT complex, which simultaneously binds cargo proteins and the ciliary motors. So far 22 subunits of the IFT complex have been identified, but insights into the biochemical architecture and especially the three-dimensional structure of this machinery are only starting to emerge because of difficulties in obtaining homogeneous material suitable for structural analysis. Here, we describe a protocol for the purification and reconstitution of a complex containing nine Chlamydomonas reinhardtii IFT proteins, commonly known as the IFT-B core complex. In our hands, this protocol routinely yields several milligrams of pure complex suitable for structural analysis by X-ray crystallography and single-particle cryo-electron microscopy.