PMID: 8962165Dec 10, 1996Paper

Crystallization and identification of an assembly defect of recombinant antenna complexes produced in transgenic tobacco plants

Proceedings of the National Academy of Sciences of the United States of America
R Flachmann, W Kühlbrandt

Abstract

A chimeric Lhcb gene encoding light-harvesting chlorophyll a/b-binding protein (LHCII) was expressed in transgenic tobacco plants. To separate native from recombinant LHCII, the protein was extended by six histidines at its C terminus. Recombinant LHCII was isolated by detergent-mediated monomerization of pure trimers followed by affinity-chromatography on Ni(2+)-NTA-agarose (NTA is nitrilotriacetic acid). Elution with imidazole yielded recombinant monomers that formed trimers readily after dilution of the detergent without further in vitro manipulations. LHCII subunits showed the typical chlorophyll a/b ratio at all steps of purification indicating no significant loss of pigments. Transgenic tobacco overexpressed amounts of recombinant protein that corresponded to about 0.7% of total LHCII. This yield suggested that expression in planta might be an alternative to the expression of eukaryotic membrane proteins in yeast. Recombinant LHCII was able to form two-dimensional crystals after addition of digalactolipids, which diffracted electrons to 3.6-A resolution. LHCII carrying a replacement of Arg-21 with Gln accumulated to only 0.004% of total thylakoid proteins. This mutant was monomeric in the photosynthetic membrane probably ...Continue Reading

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Citations

Dec 14, 2011·Photosynthesis Research·Hao PanMin Chen
Aug 1, 1997·Current Opinion in Structural Biology·J B HeymannA Engel
Feb 15, 2000·European Journal of Biochemistry·K KosemundH Paulsen
Dec 19, 2002·Critical Reviews in Biotechnology·Robert B van HuysteeBao Lige
Sep 11, 2004·Protein Expression and Purification·David M MuellerJohn E Walker
Nov 17, 2001·BMC Plant Biology·L L EgginkJ K Hoober
Sep 25, 1999·The Journal of Biological Chemistry·H WuS T Crooke
Nov 5, 1997·Protein Expression and Purification·R Grisshammer, J Tucker
Oct 14, 2006·Science·Zhiyong TangNicholas A Kotov

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