Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii

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

Research output: Contribution to journalArticle

24 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 languageEnglish
Pages (from-to)51-60
Number of pages10
JournalPhotosynthesis Research
Volume96
Issue number1
DOIs
Publication statusPublished - Apr 2008

Fingerprint

nitrilotriacetic acid
Nitrilotriacetic Acid
Photosystem I Protein Complex
Chlamydomonas reinhardtii
Thylakoids
photosystem I
histidine
Histidine
thylakoids
Pigments
rapid methods
detergents
Detergents
Purification
Paramagnetic resonance
Epitopes
spectroscopy
Assays
Spectrum Analysis
Fluorescence

Keywords

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

ASJC Scopus subject areas

  • Plant Science

Cite this

Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii. / Gulis, Galina; Narasimhulu, Kuppala V.; Fox, Lisa N.; Redding, Kevin Edward.

In: Photosynthesis Research, Vol. 96, No. 1, 04.2008, p. 51-60.

Research output: Contribution to journalArticle

Gulis, Galina ; Narasimhulu, Kuppala V. ; Fox, Lisa N. ; Redding, Kevin Edward. / Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii. In: Photosynthesis Research. 2008 ; Vol. 96, No. 1. pp. 51-60.
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