Background

Cyanobacteria have been used as the preferred model systems to study the biogenesis and function of photosynthetic protein complexes for decades. The photosynthetic apparatus in cyanobacteria is very similar to eukaryotic systems (algae and plants), but cyanobacteria have all the advantages of prokaryotic models, such as fast growth and small genomes that allow easy genetic manipulation and the use of standard tools of bacterial genetics. Particularly, Synechocystis sp. PCC 6803 (hereafter Synechocystis) is established as a favorite model organism in this field, mainly because it is transformable with high efficiency and integrates DNA by functional homologous recombination. Moreover, a glucose tolerant substrain of Synechocystis, which can proliferate using carbohydrate supplement, enables studying the function of photosynthetic genes by reverse genetics (Williams, 1988).

The biogenesis of photosystem II (PSII), a large membrane embedded pigment-protein complex responsible for photosynthetic water oxidation, has been studied intensively in Synechocystis (Nixon et al., 2010). The assembly of the PSII complex from individual subunits requires complicated machinery of many auxiliary (assembly) factors, which are typically involved in a certain assembly step (Komenda et al., 2012). A conventional approach for the analysis of PSII assembly complexes is using Synechocystis mutants lacking one or several PSII subunits. PSII biogenesis is then blocked at a particular step where the missing subunit would bind to an intermediate complex of the assembly process. In such mutants, the otherwise transient PSII pre-complexes can accumulate to a level that allows their detection and even purification, which has traditionally been achieved via His-tagged PSII (core) subunits (Dobáková et al., 2007 and 2009; Boehm et al., 2011 and 2012).

His-tag has several advantages: it is small enough to be easily introduced via a simple primer design, and it is relatively inexpensive to use. However, after His-affinity purification the preparations from Synechocystis often contain prominent contaminating proteins (Boehm et al., 2011 and 2012; Liu et al., 2011); therefore, additional purification steps are usually required to obtain the satisfactory quality for functional and structural studies. Additional purification steps may, however, compromise the yield and nativity of the complex of interest. Moreover, the levels of many protein complexes in the cell are simply too low for feasible multi-step purification protocols, PSII assembly intermediates are such an example. For these reasons, a fast, gentle, single-step purification method is highly desirable.

Affinity tags, based on biomolecular interactions offer superior specificity as compared to the general metal affinity of His-tag (Lichty et al., 2005). Large, proteinaceous tags such as small ubiquitin-like modifier (SUMO) or maltose binding protein (MBP) are well suited for the isolation of small soluble proteins due to their stable nature, which can even promote the solubility of the protein (Esposito and Chatterjee, 2006). However, for studying larger and potentially labile membrane-protein complexes, a less bulky tag with minimal interference on protein-protein interactions is preferable. Short peptide tags with high-affinity to proteins or antibodies seem to be the best suited for this purpose. One such tag is the short octapeptide called FLAG (DYKDDDDK; Hopp et al., 1988). Compared to His-, and several other tags, using FLAG-tag results in superior purity with little compromise in yield (Lichty et al., 2005). Moreover, because the elution of FLAG-tagged proteins is possible using a synthetic FLAG-peptide, the usage of disrupting chemicals, high ion concentrations or extreme pH can be avoided. Even though the price per mg purified protein of FLAG-affinity resin is approximately 50 times higher than that of Ni-NTA resin (Lichty et al., 2005); for the purification of labile and/or low-abundant complexes it can well worth the time and costs saved compared to optimizing multi-step purification procedures. Recently, FLAG-tag based approaches have been employed to purify pathogenic variants of the human Huntingtin protein from mammalian and insect cell lines (Harding et al., 2009), as well as the integral membrane Vo-part of the Pichia pastoris V-type ATPase (Li et al., 2017).

The FLAG-affinity purification protocol presented here is designed for the purification of proteins tagged with 3x FLAG epitope (DYKDDDDKDYKDDDDKDYKDDDDK). The protocol has been successfully used to resolve the roles of the PSII assembly factors Ycf39 (Knoppová et al., 2014), CyanoP (Knoppová et al., 2016), Pam68 (Bučinská et al., 2018) and RubA (Kiss et al., 2019); as well as pigment-protein complexes containing the chlorophyll-synthase (Chidgey et al., 2014), protoporphyrinogen oxidase (Skotnicová et al., 2018) and ferrochelatase enzymes (Pazderník et al., 2019). Although the method was originally developed for membrane-bound protein complexes, with minor modifications it can also be applied to soluble proteins.