Reconstitution of the spinach oxygen-evolving complex with recombinant Arabidopsis manganese-stabilizing protein.
The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII). Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from Arabidopsis thaliana. Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration. In cells grown under weak aeration, the mature protein accumulated upon induction. In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased. Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor. Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O2 evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein. We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217-227).
Betts
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SD
Department of Biology, University of Michigan, Ann Arbor 48109.
Hachigian
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Pichersky
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Yocum
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CF
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D002479:Inclusion Bodies / Q000378:metabolism; D008667:Metalloproteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D011498:Protein Precursors / Q000096:biosynthesis; D011506:Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D018724:Spinacia oleracea / Q000737:chemistry*
D001426:Bacterial Proteins; D018076:DNA, Complementary; D008667:Metalloproteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D011498:Protein Precursors; D011506:Proteins; D011994:Recombinant Proteins; C064388:oxygen-evolving complex, 33 kDa protein; C054266:photosystem II manganese-stabilizing protein; D010100:Oxygen