Abstract
During storage in the silk gland, the N-terminal domain (NT) of spider silk proteins (spidroins) keeps the aggregation-prone repetitive region in solution at extreme concentrations. We observe that NTs from different spidroins have co-evolved with their respective repeat region, and now use an NT that is distantly related to previously used NTs, for efficient recombinant production of the amyloid-β peptide (Aβ) implicated in Alzheimer’s disease. A designed variant of NT from Nephila clavipes flagelliform spidroin, which in nature allows production and storage of β-hairpin repeat segments, gives exceptionally high yields of different human Aβ variants as a solubility tag. This tool enables efficient production of target peptides also in minimal medium and gives up to 10 times more isotope-labeled monomeric Aβ peptides per liter bacterial culture than previously reported.
Highlights
Orb-weaving spiders manufacture up to seven different silks, e.g. dragline silk derived from major ampullate silk proteins and flagelliform silk derived from flagelliform spidroins (FlSp)
A phylogenetic tree based on sequence alignment of 67 NTs found in GenBank (Supplementary Fig. S1) reveal evolutionary relationships between NT and their respective repetitive regions (Fig. 1A)
The NTs of different spidroin types, which are defined by the nature of their respective repetitive regions, have been conserved through evolution of different spider species
Summary
Orb-weaving spiders manufacture up to seven different silks, e.g. dragline silk derived from major ampullate silk proteins (spidroins, MaSp) and flagelliform silk derived from flagelliform spidroins (FlSp). The divergent and large aggregation-prone repetitive regions of the spidroins determine the mechanical properties of the respective spider silks, while the terminal domains regulate silk fiber formation[2,3]. Despite their high aggregation propensity the spidroins can be stored at extremely high concentrations (30–50% w/v) in the spider silk gland, solubilized by the NT domain[1,4]. These proteins/peptides often exhibit high β-sheet propensity, which make them prone to aggregate and form insoluble amyloid fibrils[11] These intrinsic properties of amyloid-forming proteins make high-yield biochemical production challenging, yet the availability of pure protein samples is crucial for studying protein self-assembly and its associated neurotoxicity in vitro and in vivo. We describe a useful solubility tag for production of aggregation-prone proteins and peptides, and demonstrate that this tool enables very efficient production of native and isotope-labeled Aβ peptides
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