A way to identify archaellins in Halobacterium salinarum archaella by FLAG-tagging

Abstract

In the current study, haloarchaea Halobacterium salinarum cells were transformed individually with each of the modified archaellin genes (flaA1, flaA2 and flaB2) containing an oligonucleotide insert encoding the FLAG peptide (DYKDDDDK). The insertion site was selected to expose the FLAG peptide on the archaella filament surface. Three types of transformed cells synthesizing archaella, containing A1, A2, or B2 archaellin modified with FLAG peptide were obtained. Electron microscopy of archaella has demonstrated that in each case the FLAG peptide is available for the specific antibody binding. It was shown for the first time that the B2 archaellin, like archaellins A1 and A2, is found along the whole filament length.

Homeostasis of metabolites in Escherichia coli on transition from anaerobic to aerobic conditions and the transient secretion of pyruvate

“…Although there are many reports of FLAG-tagged bacterial proteins, most of these use the tagging for pull-downs or western blotting. We have only identified a small number of publications in which FLAG tags have been used to localize proteins within bacteria, either using immunofluorescence [37][38][39] or nanogold particles [40]. As in our case the change in localization is likely to be rather small and transient, and as fluorescent labelling had previously been unable to distinguish between the two, we opted for nanogold labelling.…”

Effects of N-Glycosylation Site Removal in Archaellins on the Assembly and Function of Archaella in Methanococcus maripaludis

“…In addition, sequences in the internal hypervariable region of bacterial flagellin can be replaced with completely unrelated sequences [ 73 ]. This is also true for archaellins in Halobacterium salinarum where both FLAG (8 amino acid peptide) and a gold-binding 12 amino acid peptide have been inserted into variable regions of different archaellins and these mutant proteins were still able to be assembled into archaella [ 74 ]. However, for type IV pilins, it has been shown in a number of studies that very small changes at key amino acids in the major pilins can result in instability of the pilins and ones that cannot assemble into pili [ 75 – 77 ].…”

Flagellar filament bio-templated inorganic oxide materials – towards an efficient lithium battery anode

“…Halophilic archaea are safe to humans and quite un-demanding in terms of growth environment (advantageously, requiring a high salt concentration), and modified flagella can be isolated and purified easily with yields up to 20 mg L −1 (compared to 10 mg L −1 in the case of M13 viruses). Previously, we have demonstrated the use of flagella of halophilic archaea species Halobacterium salinarum as a biotemplate for the selective binding of target substances 21 26 . H. salinarum flagellar filaments are formed from five homologous protein subunits-flagellins FlgA1, A2, B1, B2, B3 in which FlgA1 and A2 are the major components which are distributed along the full filament length 26 27 .…”

“…Previously, we have demonstrated the use of flagella of halophilic archaea species Halobacterium salinarum as a biotemplate for the selective binding of target substances 21 26 . H. salinarum flagellar filaments are formed from five homologous protein subunits-flagellins FlgA1, A2, B1, B2, B3 in which FlgA1 and A2 are the major components which are distributed along the full filament length 26 27 . To create binding sites for templating purposes, genetically modified strains can be constructed 21 26 such that FlgA1 and A2 are modified with an inserted FLAG peptide (DYKDDDDK) sequence 21 26 .…”

“…H. salinarum flagellar filaments are formed from five homologous protein subunits-flagellins FlgA1, A2, B1, B2, B3 in which FlgA1 and A2 are the major components which are distributed along the full filament length 26 27 . To create binding sites for templating purposes, genetically modified strains can be constructed 21 26 such that FlgA1 and A2 are modified with an inserted FLAG peptide (DYKDDDDK) sequence 21 26 . The carboxyl groups of aspartate and glutamate side chains are good sites for positively charged ion binding.…”