This chapter describes purification procedures for a set of fusion proteins containing the first 22 amino acids of yeast cytochrome oxidase subunit IV (COXIV) fused to the amino terminus of mouse dihydrofolate reductase (DHFR) through a three-amino acid linker. The isolation of pure precursor protein takes advantage of the fact that the folate analog methotrexate binds tightly to DHFR. Methotrexate can therefore be used as a ligand for affinity chromatography of these precursor proteins directly from cell extracts. However, point mutations that lower the affinity of DHFR for the ligand also lower the effectiveness of this purification step. The bound COXIV-DHFR can be eluted with an excess of the natural ligand, dihydrofolate. However, this eluate still contains several contaminating proteins. In a second step, COXIV-DHFR is purified to homogeneity by anion exchange chromatography. The protein binds to the support by the presence of several positively charged amino acids in the COXIV pre-sequence. All other contaminants in the eluate from the affinity column are found in the flow-through fraction. The precursor is then eluted in the presence of high ionic strength. The chapter describes utilization of purified precursors in the study of mitochondrial protein import. Purified precursor proteins are used to establish their structural features that are required for import competence.
ASJC Scopus subject areas
- Cell Biology