Purification of IFT particle proteins and preparation of recombinant proteins for structural and functional analysis.
Intraflagellar transport (IFT) is characterized by a robust bidirectional movement of large proteinaceous particles along the length of eukaryotic cilia and flagella. Essential for the assembly and function of the organelle, IFT is believed to transport a large array of ciliary components in and out of the organelle. Biochemical analysis of the proteins involved with this transport has been largely dependent on the ability to isolate suitable quantities of intact cilia or flagella. One model organism, Chlamydomonas reinhardtii, has proven to be especially well-suited for such endeavors. Indeed, many of the IFT particle proteins were initially identified through biochemical analysis of green algae. This chapter describes some of the most effective methods for the purification of IFT particle proteins from Chlamydomonas flagella. This chapter also describes complementary approaches where recombinant IFT proteins are generated with affinity tags that allow rapid and specific purification. The recombinant proteins can be used to analyze protein-protein interactions and can be directly delivered to mutant cells to analyze functional domains. Although the techniques described here are focused entirely on Chlamydomonas IFT proteins, the approaches, especially regarding recombinant proteins, should be applicable to the study of IFT machinery in other model organisms.
Behal
Robert H
RH
Department of Microbiology, Molecular Biology and Biochemistry and the Center for Reproductive Biology,LSS142, University of Idaho, Moscow, Idaho 83844-3052, USA.
Betleja
Ewelina
E
Cole
Douglas G
DG
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002499:Centrifugation, Density Gradient / Q000295:instrumentation / Q000379:methods; D016825:Chlamydomonas reinhardtii / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D005407:Flagella / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D015800:Protozoan Proteins; D011994:Recombinant Proteins