Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii

Galina Gulis; Kuppala V. Narasimhulu; Lisa N. Fox; Kevin Edward Redding

doi:10.1007/s11120-007-9283-9

Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii

Galina Gulis, Kuppala V. Narasimhulu, Lisa N. Fox, Kevin Edward Redding

Arizona State University

Research output: Contribution to journal › Article

27 Citations (Scopus)

Abstract

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.

Original language	English
Pages (from-to)	51-60
Number of pages	10
Journal	Photosynthesis Research
Volume	96
Issue number	1
DOIs	https://doi.org/10.1007/s11120-007-9283-9
Publication status	Published - Apr 2008

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Keywords

Chlamydomonas reinhardtii
Epitope tagging
His-tag
Membrane protein
Photosystem 1
Reaction center

ASJC Scopus subject areas

Plant Science

Cite this

Gulis, G., Narasimhulu, K. V., Fox, L. N., & Redding, K. E. (2008). Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii. Photosynthesis Research, 96(1), 51-60. https://doi.org/10.1007/s11120-007-9283-9

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abstract = "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.",

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AB - 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.

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10.1007/s11120-007-9283-9