Purification and characterization of photosystem I complex from Synechocystis sp. PCC 6803 by expressing histidine-tagged subunits.
We generated Synechocystis sp. PCC 6803 strains, designated F-His and J-His, which express histidine-tagged PsaF and PsaJ subunits, respectively, for simple purification of the photosystem I (PSI) complex. Six histidine residues were genetically added to the C-terminus of the PsaF subunit in F-His cells and the N-terminus of the PsaJ subunit in J-His cells. The histidine residues introduced had no apparent effect on photoautotrophic growth of the cells or the activity of PSI and PSII in thylakoid membranes. PSI complexes could be simply purified from the F-His and J-His cells by Ni2+-affinity column chromatography. When thylakoid membranes corresponding to 20 mg chlorophyll were used, PSI complexes corresponding to about 7 mg chlorophyll could be purified in both strains. The purified PSI complexes could be separated into monomers and trimers by ultracentrifugation in glycerol density gradient and high activity was recorded for trimers isolated from the F-His and J-His strains. Blue-Native PAGE and SDS-PAGE analysis of monomers and trimers indicated the existence of two distinct monomers with different subunit compositions and no contamination of PSI with other complexes, such as PSII and Cyt b(6)f. Further analysis of proteins and lipids in the purified PSI indicated the presence of novel proteins in the monomers and about six lipid molecules per monomer unit in the trimers. These results demonstrate that active PSI complexes can be simply purified from the constructed strains and the strains are very useful tools for analysis of PSI.
Kubota
Hisako
H
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan.
Sakurai
Isamu
I
Katayama
Kenta
K
Mizusawa
Naoki
N
Ohashi
Shunsuke
S
Kobayashi
Masami
M
Zhang
Pengpeng
P
Aro
Eva-Mari
EM
Wada
Hajime
H
D001483:Base Sequence; D017931:DNA Primers; D005288:Ferredoxins / Q000378:metabolism; D005798:Genes, Bacterial; D006639:Histidine / Q000032:analysis; D008055:Lipids / Q000302:isolation & purification; D009842:Oligopeptides / Q000032:analysis; D010101:Oxygen Consumption; D045331:Photosystem I Protein Complex / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D045332:Photosystem II Protein Complex / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010858:Pigmentation; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D046939:Synechocystis / Q000235:genetics* / Q000378:metabolism; D020524:Thylakoids / Q000378:metabolism / Q000648:ultrastructure
D017931:DNA Primers; D005288:Ferredoxins; C471213:His-His-His-His-His-His; D008055:Lipids; D009842:Oligopeptides; D045331:Photosystem I Protein Complex; D045332:Photosystem II Protein Complex; D021122:Protein Subunits; D006639:Histidine