Journal
PHOTOSYNTHESIS RESEARCH
Volume 96, Issue 1, Pages 51-60Publisher
SPRINGER
DOI: 10.1007/s11120-007-9283-9
Keywords
His-tag; epitope tagging; membrane protein; reaction center; photosystem 1; chlamydomonas reinhardtii
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Funding
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [0854851] Funding Source: National Science Foundation
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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 His(6)-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 His(6)-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.
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