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Planta

, Volume 181, Issue 2, pp 204–211 | Cite as

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

  • H. Paulsen
  • U. Rümler
  • W. Rüdiger
Article

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.

Key words

Antenna complex Expression plasmids Light-harvesting chlorophyll a/b-binding protein Pigment-protein assembly Pisum (light-harvesting complex) Thylakoid biogenesis 

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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Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • H. Paulsen
    • 1
  • U. Rümler
    • 1
  • W. Rüdiger
    • 1
  1. 1.Botanisches Institut III der UniversitätMünchen 19Germany

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