Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii

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

Research output: Contribution to journal › Article › peer-review

31 Scopus citations

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 (US)
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
State	Published - Apr 2008
Externally published	Yes

Keywords

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

ASJC Scopus subject areas

Biochemistry
Plant Science
Cell Biology

Access to Document

10.1007/s11120-007-9283-9

Cite this

@article{38db62c870cb4bdc9b2231a535c6bef1,

title = "Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii",

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.",

keywords = "Chlamydomonas reinhardtii, Epitope tagging, His-tag, Membrane protein, Photosystem 1, Reaction center",

author = "Galina Gulis and Narasimhulu, \{Kuppala V.\} and Fox, \{Lisa N.\} and Redding, \{Kevin E.\}",

note = "Funding Information: Acknowledgments 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 photo-bleaching measurements. This work was supported by an NSF CAREER award (MCB-0347935) to Kevin E. Redding.",

year = "2008",

month = apr,

doi = "10.1007/s11120-007-9283-9",

language = "English (US)",

volume = "96",

pages = "51--60",

journal = "Photosynthesis research",

issn = "0166-8595",

publisher = "Springer",

number = "1",

}

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 E.

N1 - Funding Information: Acknowledgments 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 photo-bleaching measurements. This work was supported by an NSF CAREER award (MCB-0347935) to Kevin E. Redding.

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 - https://www.scopus.com/pages/publications/39849111322

UR - https://www.scopus.com/pages/publications/39849111322#tab=citedBy

U2 - 10.1007/s11120-007-9283-9

DO - 10.1007/s11120-007-9283-9

M3 - Article

C2 - 18175204

AN - SCOPUS:39849111322

SN - 0166-8595

VL - 96

SP - 51

EP - 60

JO - Photosynthesis research

JF - Photosynthesis research

IS - 1

ER -