Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii.
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.
Gulis
Galina
G
Department of Chemistry, University of Alabama, 206 Shelby Hall, 250 Hackberry Lane, Tuscaloosa, AL 35487-0336, USA.
Narasimhulu
Kuppala V
KV
Fox
Lisa N
LN
Redding
Kevin E
KE
D020418:Algal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D045331:Photosystem I Protein Complex / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D020524:Thylakoids / Q000378:metabolism
D020418:Algal Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D045331:Photosystem I Protein Complex; D006639:Histidine