Centrin is a component of an assortment of calcium-modulated cytoskeletal fiber systems found associated with flagellar basal apparatus, centrosomes, and mitotic spindle poles of eukaryotic cells. It is an acidic, low-molecular-weight, calcium-binding phosphoprotein that belongs to the EF-hand superfamily of calcium-binding proteins centrin-based systems include striated flagellar roots, transition zone fibers, fibrous bundles, and pericentriolar lattice. Centrin-based fibers play a role in anchoring the flagellar basal apparatus to the nucleus, in positioning of basal bodies and centrioles during the cell cycle, in severing microtubules at the time of flagellar excision, and in coiling the paraflagellar rod of transverse flagella. In this chapter is presented detailed methods for generating milligram quantities of pure centrin. These methods involve expression of a cDNA clone encoding Chlamydomonas centrin in Escherichia coli, and chromatographic purification of the expressed protein. The chromatographic method described works well for the purification of centrin from isolated Chlamydomonas flagella. The chapter discusses the construction of E. coli BL21 pT7-5Cen, induction and lysis of E. coli BL21 pT7-5Cen, phenyl-sepharose CLdB chromatography, superose-12 gel filtration chromatography, mono Q anion-exchange chromatography, and the troubleshooting methods. Problems encountered include inactive ampicillin, which may result in the selection and growth of bacteria that have lost the pT7-Zen plasmid; failure of transcription induction because of inactive Isopropylthio-β-glactoside (IPTG) or T7 promoter; induction of protein products that are larger or smaller than centrin because of improper bacterial transcription and/or translation; inefficient digestion of the bacterial cell wall with lysozyme; and inefficient binding of centrin to a fouled phenylsepharose column. To troubleshoot and assess the efficiency of the purification procedure, it is advised to prepare SDS-PAGE samples of E. coli BL21 pT7-5Cen before and after IPTG induction, whole-cell lysate following sonication, P1, S1, P2, S2, column voids, and column elution fractions.
ASJC Scopus subject areas
- Cell Biology