Abstract
Expression and purification of recombinant proteins are important for the structure–function study of phytochromes. However, it is difficult to purify phytochrome proteins from natural sources or using a bacterial expression system, due to the presence of multiple phytochrome species and low expression and solubility, respectively. Here we describe the expression of recombinant full-length plant phytochromes in the yeast Pichia pastoris, and the spectral analysis of chromophore-assembled phytochromes before and after the purification by streptavidin affinity chromatography.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Rockwell NC, Su YS, Lagarias JC (2006) Phytochrome structure and signaling mechanisms. Annu Rev Plant Biol 57:837–858
Li J, Li G, Wang H, Wang Deng X (2011) Phytochrome signaling mechanisms. Arabidopsis Book 9:e0148
Xu X, Paik I, Zhu L, Huq E (2015) Illuminating progress in phytochrome-mediated light signaling pathways. Trends Plant Sci 20:641–650
Lapko VN, Song PS (1995) A simple and improved method of isolation and purification for native oat phytochrome. Photochem Photobiol 62:194–198
Hill C, Gartner W, Towner P, Braslavsky SE, Schaffner K (1994) Expression of phytochrome apoprotein from Avena sativa in Escherichia coli and formation of photoactive chromoproteins by assembly with phycocyanobilin. Eur J Biochem 223:69–77
Mozley D, Remberg A, Gartner W (1997) Large-scale generation of affinity-purified recombinant phytochrome chromopeptide. Photochem Photobiol 66:710–715
Murphy JT, Lagarias JC (1997) Purification and characterization of recombinant affinity peptide-tagged oat phytochrome A. Photochem Photobiol 65:750–758
Gartner W, Hill C, Worm K, Braslavsky SE, Schaffner K (1996) Influence of expression system on chromophore binding and preservation of spectral properties in recombinant phytochrome A. Eur J Biochem 236:978–983
Mukougawa K, Kanamoto H, Kobayashi T, Yokota A, Kohchi T (2006) Metabolic engineering to produce phytochromes with phytochromobilin, phycocyanobilin, or phycoerythrobilin chromophore in Escherichia coli. FEBS Lett 580:1333–1338
Wu SH, Lagarias JC (1996) The methylotrophic yeast Pichia pastoris synthesizes a functionally active chromophore precursor of the plant photoreceptor phytochrome. Proc Natl Acad Sci U S A 93:8989–8994
Shin AY, Han YJ, Song PS, Kim JI (2014) Expression of recombinant full-length plant phytochromes assembled with phytochromobilin in Pichia pastoris. FEBS Lett 588:2964–2970
Shin AY, Han YJ, Baek A, Ahn T, Kim SY, Nguyen TS, Son M, Lee KW, Shen Y, Song PS, Kim JI (2016) Evidence that phytochrome functions as a protein kinase in plant light signalling. Nat Commun 7:11545
Jeong AR, Lee SS, Han YJ, Shin AY, Baek A, Ahn T, Kim MG, Kim YS, Lee KW, Nagatani A, Kim JI (2016) New constitutively active phytochromes exhibit light-independent signaling activity. Plant Physiol 171:2826–2840
Romanos M (1995) Advances in the use of Pichia pastoris for high-level gene expression. Curr Opin Biotechnol 6:527–533
Quail PH, Briggs WR, Chory J, Hangarter RP, Harberd NP, Kendrick RE, Koornneef M, Parks B, Sharrock RA, Schafer E, Thompson WF, Whitelam GC (1994) Spotlight on phytochrome nomenclature. Plant Cell 6:468–471
Terry MJ, Maines MD, Lagarias JC (1993) Inactivation of phytochrome- and phycobiliprotein-chromophore precursors by rat liver biliverdin reductase. J Biol Chem 268:26099–26106
Acknowledgments
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (grant no. 2017R1A2B4010349) and in part by Next-Generation BioGreen 21 Program from Rural Development Administration, Republic of Korea (grant no. PJ01332701).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Han, YJ., Cho, JY., Kim, JI. (2019). Expression, Purification, and Spectral Characterization of Phytochromes. In: Hiltbrunner, A. (eds) Phytochromes. Methods in Molecular Biology, vol 2026. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9612-4_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9612-4_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9611-7
Online ISBN: 978-1-4939-9612-4
eBook Packages: Springer Protocols