Exploring the structure and function of protein complexes requires their isolation in the native state-a task that is made challenging when studying labile and/or low abundant complexes. The difficulties in preparing membrane-protein complexes are especially notorious. The cyanobacterium Synechocystis sp. PCC 6803 is a widely used model organism for the physiology of oxygenic phototrophs, and the biogenesis of membrane-bound photosynthetic complexes has traditionally been studied using this cyanobacterium. In a typical approach, the protein complexes are purified with a combination of His-affinity chromatography and a size-based fractionation method such as gradient ultracentrifugation and/or native electrophoresis. However, His-affinity purification harbors prominent contaminants and the levels of many proteins are too low for a feasible multi-step purification. Here, we have developed a purification method for the isolation of 3x FLAG-tagged proteins from the membrane and soluble fractions of Synechocystis. Soluble proteins or solubilized thylakoids are subjected to a single affinity purification step that utilizes the highly specific binding of FLAG-affinity resin. After an intensive wash, the captured proteins are released from the resin under native conditions using an excess of synthetic 3x FLAG peptide. The protocol allows fast isolation of low abundant protein complexes with a superb purity.

Centre Algatech Institute of Microbiology Czech Academy of Sciences Třeboň Czech Republic

Faculty of Science University of South Bohemia České Budějovice 370 05 Czech Republic

Zobrazit více v PubMed

Boehm M., Romero E., Reisinger V., Yu J., Komenda J., Eichacker L. A., Dekker J. P. and Nixon P. J.(2011). Investigating the early stages of photosystem II assembly in Synechocystis sp. PCC 6803: isolation of CP47 and CP43 complexes . J Biol Chem 286(17): 14812-14819. PubMed PMC

Boehm M., Yu J., Reisinger V., Bečková M., Eichacker L. A., Schlodder E., Komenda J. and Nixon P. J.(2012). Subunit composition of CP43-less photosystem II complexes of Synechocystis sp. PCC 6803: implications for the assembly and repair of photosystem II . Philos Trans R Soc Lond B Biol Sci 367(1608): 3444-3454. PubMed PMC

Bučinská L., Kiss É., Koník P., Knoppová J., Komenda J. and Sobotka R.(2018). The ribosome-bound protein Pam68 promotes insertion of chlorophyll into the CP47 subunit of photosystem II. Plant Physiol 176(4): 2931-2942. PubMed PMC

Chidgey J. W., Linhartová M., Komenda J., Jackson P. J., Dickman M. J., Canniffe D. P., Konik P., Pilny J., Hunter C. N. and Sobotka R.(2014). A cyanobacterial chlorophyll synthase-HliD complex associates with the Ycf39 protein and the YidC/Alb3 insertase. Plant Cell 26(3): 1267-1279. PubMed PMC

Dobáková M., Sobotka R., Tichý M. and Komenda J.(2009). Psb28 protein is involved in the biogenesis of the photosystem II inner antenna CP47(PsbB) in the cyanobacterium Synechocystis sp. PCC 6803 . Plant Physiol 149(2): 1076-1086. PubMed PMC

Dobáková M., Tichý M. and Komenda J.(2007). Role of the PsbI protein in photosystem II assembly and repair in the cyanobacterium Synechocystis sp. PCC 6803 . Plant Physiol 145(4): 1681-1691. PubMed PMC

Esposito D. and Chatterjee D. K.(2006). Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17(4): 353-358. PubMed

Harding R. J., Loppnau P., Ackloo S., Lemak A., Hutchinson A., Hunt B., Holehouse A. S., Ho J. C., Fan L., Toledo-Sherman L., Seitova A. and Arrowsmith C. H.(2019). Design and characterization of mutant and wildtype huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems. J Biol Chem 294(17): 6986-7001. PubMed PMC

Hopp T. P., Prickett K. S., Price V. L., Libby R. T., March C. J., Cerretti D. P., Urdal D. L. and Conlon P. J.(1988). A short polypeptide marker sequence useful for recombinant protein identification and purification. Nature Biotechnology 6: 1204-1210.

Kiss É., Knoppová J., Aznar G. P., Pilný J., Yu J., Halada P., Nixon P. J., Sobotka R. and Komenda J.(2019). A Photosynthesis-Specific Rubredoxin-Like Protein Is Required for Efficient Association of the D1 and D2 Proteins during the Initial Steps of Photosystem II Assembly. Plant Cell 31(9): 2241-2258. PubMed PMC

Knoppová J., Sobotka R., Tichý M., Yu J., Konik P., Halada P., Nixon P. J. and Komenda J.(2014). Discovery of a chlorophyll binding protein complex involved in the early steps of photosystem II assembly in Synechocystis . Plant Cell 26(3): 1200-1212. PubMed PMC

Knoppová J., Yu J., Konik P., Nixon P. J. and Komenda J.(2016). CyanoP is involved in the early steps of Photosystem II assembly in the cyanobacterium Synechocystis sp. PCC 6803 . Plant Cell Physiol 57(9): 1921-1931. PubMed

Komenda J., Sobotka R. and Nixon P. J.(2012). Assembling and maintaining the Photosystem II complex in chloroplasts and cyanobacteria. Curr Opin Plant Biol 15(3): 245-251. PubMed

Li S., Hong T., Wang K., Lu Y. and Zhou M.(2017). Dissociation and purification of the endogenous membrane-bound Vo complex from Pichia pastoris. Protein Expr Purif 138: 76-80. PubMed

Lichty J. J., Malecki J. L., Agnew H. D., Michelson-Horowitz D. J. and Tan S.(2005). Comparison of affinity tags for protein purification. Protein Expr Purif 41(1): 98-105. PubMed

Liu H., Roose J. L., Cameron J. C. and Pakrasi H. B.(2011). A genetically tagged Psb27 protein allows purification of two consecutive photosystem II(PSII) assembly intermediates in Synechocystis 6803, a cyanobacterium . J Biol Chem 286(28): 24865-24871. PubMed PMC

Nixon P. J., Michoux F., Yu J., Boehm M. and Komenda J.(2010). Recent advances in understanding the assembly and repair of photosystem II. Ann Bot 106(1): 1-16. PubMed PMC

Pazderník M., Mareš J., Pilný J. and Sobotka R.(2019). The antenna-like domain of the cyanobacterial ferrochelatase can bind chlorophyll and carotenoids in an energy-dissipative configuration. J Biol Chem 294(29): 11131-11143. PubMed PMC

Porra R. J., Thompson W. A. and Kriedemann P. E.(1989). Determination og accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy . Biochimica et Biophysica Acta 975: 384-394.

Rippka R., Deruelles J., Waterbury J. B., Herdman M. and Stanier R. Y.(1979). Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. Microbiology 111(1): 1-61.

Skotnicová P., Sobotka R., Shepherd M., Hajek J., Hrouzek P. and Tichy M.(2018). The cyanobacterial protoporphyrinogen oxidase HemJ is a new b-type heme protein functionally coupled with coproporphyrinogen III oxidase. J Biol Chem 293(32): 12394-12404. PubMed PMC

Williams J. G. K.(1988). Construction of specific mutations in photosystem II photosynthetic reaction center by genetic-engineering methods in Synechocystis 6803 . Methods in Enzymology 167: 766-778.

Chlorophyll biosynthesis under the control of arginine metabolism

FtsH4 protease controls biogenesis of the PSII complex by dual regulation of high light-inducible proteins

Assembly of D1/D2 complexes of photosystem II: Binding of pigments and a network of auxiliary proteins

Psb34 protein modulates binding of high-light-inducible proteins to CP47-containing photosystem II assembly intermediates in the cyanobacterium Synechocystis sp. PCC 6803