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Reconstitution of pigment-containing complexes from light-harvesting chlorophyll a/b-binding protein overexpressed inEscherichia coli

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Abstract

A gene for a light-harvesting chlorophyll (Chl) a/b-binding protein (LHCP) from pea (Pisum sativum L.) has been cloned in a bacterial expression vector. Bacteria (Escherichia coli) transformed with this construct produced up to 20% of their protein as pLHCP, a derivative of the authentic precursor protein coded for by the pea gene with three amino-terminal amino acids added and-or exchanged, or as a truncated LHCP carrying a short amino-terminal deletion into the mature protein sequence. Following the procedure of Plumley and Schmidt (1987, Proc. Natl. Acad. Sci. USA84, 146–150), all bacteria-produced LHCP derivatives can be reconstituted with acetone extracts from pea thylakoids or with isolated pigments to yield pigment-protein complexes that are stable during partially denaturing polyacrylamide-gel electrophoresis. The spectroscopic properties of these complexes closely resemble those of the light-harvesting complex associated with photosystem II (LHCII) isolated from pea thylakoids. The pigment requirement for the reconstitution is highly specific for the pigments found in native LHCII: Chl a and b as well as at least two out of three xanthophylls are necessary. Varying the Chl a:Chl b ratios in the reconstitution mixtures changes the yields of complex formed but not the Chl a:Chl b ratio in the complex. We conclude that LHCP-pigment assembly in vitro is highly specific and that the complexes formed are structurally similar to LHCII. The N-terminal region of the protein can be varied without affecting complex formation and therefore does not seem to be involved in pigment binding.

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Abbreviations

CD:

circular dichrosim

Chl:

chlorophyll

IPTG:

isopropyl thiogalactoside

LDS:

lithium dodecyl sulfate

LHCII:

light-harvesting complex associated with photosystem II

LHCP:

light-harvesting chlorophyll a/b-binding protein

LHCPX:

LHCP derivatives overproduced in bacteria

pLHCP:

precursor of light-harvesting Chl a/b-binding protein

SDS:

sodium dodecyl sulfate

Tris:

2-amino-2-(hydroxymethyl)-1,3-propanediol

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Authors and Affiliations

  1. Botanisches Institut III der Universität, Menzinger Strasse 67, D-8000, München 19, Germany

    H. Paulsen, U. Rümler & W. Rüdiger

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  1. H. Paulsen
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  2. U. Rümler
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  3. W. Rüdiger
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Dedicated to Professor Hans Mohr on the occasion of his 60th birthday

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Paulsen, H., Rümler, U. & Rüdiger, W. Reconstitution of pigment-containing complexes from light-harvesting chlorophyll a/b-binding protein overexpressed inEscherichia coli . Planta 181, 204–211 (1990). https://doi.org/10.1007/BF02411539

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