Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii
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We have developed a rapid method for isolation of the Photosystem I (PS1) complex from Chlamydomonas reinhardtii using epitope tagging. Six histidine residues were genetically added to the N-terminus of the PsaA core subunit of PS1. The His6-tagged PS1 could be purified with a yield of 80–90% from detergent-solubilized thylakoid membranes within 3 h in a single step using a Ni-nitrilotriacetic acid (Ni-NTA) column. Immunoblots and low-temperature fluorescence analysis indicated that the His6-tagged PS1 preparation was highly pure and extremely low in uncoupled pigments. Moreover, the introduced tag appeared to have no adverse effect upon PS1 structure/function, as judged by photochemical assays and EPR spectroscopy of isolated particles, as well as photosynthetic growth tests of the tagged strain.
KeywordsHis-tag Epitope tagging Membrane protein Reaction center Photosystem 1 Chlamydomonas reinhardtii
The authors wish to thank Erica Livingston and Lynn Thronson for their contributions to the early stages of the cloning, and Fabrice Rappaport for assistance with the P700 photobleaching measurements. This work was supported by an NSF CAREER award (MCB-0347935) to Kevin E. Redding.
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