Reconstitution of pigment-containing complexes from light-harvesting chlorophyll a/b-binding protein overexpressed inEscherichia coli |
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Authors: | H Paulsen U Rümler W Rüdiger |
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Institution: | 1. Botanisches Institut III der Universit?t, Menzinger Strasse 67, D-8000, München 19, Germany
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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.
Dedicated to Professor Hans Mohr on the occasion of his 60th birthday |
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Keywords: | Antenna complex Expression plasmids Light-harvesting chlorophyll a/b-binding protein Pigment-protein assembly Pisum (light-harvesting complex) Thylakoid biogenesis |
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