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. 1993 Mar 15;90(6):2155–2159. doi: 10.1073/pnas.90.6.2155

Expression of the functional mature chloroplast triose phosphate translocator in yeast internal membranes and purification of the histidine-tagged protein by a single metal-affinity chromatography step.

B Loddenkötter 1, B Kammerer 1, K Fischer 1, U I Flügge 1
PMCID: PMC46044  PMID: 11607374

Abstract

The mature part of the chloroplast triose phosphate-phosphate translocator was cloned into the yeast expression vector pEVP11. This construct was used to transform cells from both Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. The chloroplast translocator protein was functionally expressed in the transformed yeast cells and represented about 1-2% of the Sch. pombe cell membrane protein. It was localized to mitochondrial membranes and/or membranes of the rough endoplasmic reticulum. In order to purify the recombinant translocator protein, a sequence encoding a C-terminal tag of six histidine residues was introduced into the corresponding cDNA. The expressed histidine-tagged translocator protein was purified from the transformed yeast cells under nondenaturing conditions to apparent homogeneity by a single-step affinity chromatography using a Ni2+. nitrilotriacetic acid resin. Both the expressed triose phosphate translocator and the recombinant histidine-tagged protein possess substrate specificities identical to those of the authentic chloroplast protein, providing definitive evidence for its identity as the triose phosphate translocator and further disproving its assignment as the receptor for chloroplast protein import. The yeast expression system in combination with the Ni2+. nitrilotriacetic acid chromatography thus provides a valuable tool for the production of purified membrane proteins in a functional state.

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Selected References

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