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 | |
| 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
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 journal › Article
}
TY - JOUR
T1 - Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii
AU - Gulis, Galina
AU - Narasimhulu, Kuppala V.
AU - Fox, Lisa N.
AU - Redding, Kevin Edward
PY - 2008/4
Y1 - 2008/4
N2 - 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.
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.
KW - Chlamydomonas reinhardtii
KW - Epitope tagging
KW - His-tag
KW - Membrane protein
KW - Photosystem 1
KW - Reaction center
UR - http://www.scopus.com/inward/record.url?scp=39849111322&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=39849111322&partnerID=8YFLogxK
U2 - 10.1007/s11120-007-9283-9
DO - 10.1007/s11120-007-9283-9
M3 - Article
C2 - 18175204
AN - SCOPUS:39849111322
VL - 96
SP - 51
EP - 60
JO - Photosynthesis Research
JF - Photosynthesis Research
SN - 0166-8595
IS - 1
ER -