Efficient protein production inspired by how spiders make silk

Kronqvist, Nina; Sarr, Médoune; Lindqvist, Anton; Nordling, Kerstin; Otikovs, Mārtiņš; Venturi, Luca; Pioselli, Barbara; Purhonen, Pasi; Landreh, Michael; Biverstål, Henrik; Toleikis, Zigmantas; Sjöberg, Lisa; Pelizzi, Nicola; Jörnvall, Hans; Hebert, Hans; Jaudzems, Kristaps; Curstedt, Tore; Rising, Anna; Johansson, Jan

doi:10.1038/ncomms15504

Cited by 111 publications

(161 citation statements)

References 80 publications

(110 reference statements)

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“…To prevent dimerization of NT FlSp at low pH we introduced the D40K and K65D mutations 6 in NT FlSp from Nephila clavipes (Nc) ( Fig. 1B) (numbering as described previously 6 , wherefore the mutations correspond to positions 36 and 60 in Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

High-yield Production of Amyloid-β Peptide Enabled by a Customized Spider Silk Domain

Abelein

Chen

Aleksis

et al. 2020

Sci Rep

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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.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 various spidroins share a common architecture -a large core repetitive region capped by globular N-and C-terminal domains (NT and CT) 1 . 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 .The NT dimerizes upon a drop in pH, which is crucial for silk fiber formation 1,5 . To ensure solubility also at low pH and widen the applicability of NT as a solubility enhancing fusion partner, a charged-reversed mutant has been designed (referred to as NT* MaSp ) 6 . The previously reported NT* MaSp tag is derived from the NT domain of Euprosthenops australis MaSp1 and folds as a five-helix bundle 6,7 . NT* MaSp is a pH insensitive constitutive monomer, highly stable and extremely soluble, and has been successfully applied for efficient production and purification of, among others, lung surfactant protein analogs, cholecystokinin-58, human antimicrobial cathelicidin and a designed β-sheet protein 6,8 .Aggregation-prone proteins and peptides are associated with several neurodegenerative disorders, e.g. Alzheimer's disease (AD), the most prevalent form of dementia 9,10 . 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. This is probably one important reason behind the fact that, despite immense efforts, the exact mechanisms of Aβ self-assembly are still unknown 9-11 . Recent advances have howe...

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Section: Resultsmentioning

confidence: 99%

High-yield Production of Amyloid-β Peptide Enabled by a Customized Spider Silk Domain

Abelein

Chen

Aleksis

et al. 2020

Sci Rep

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“…For generating rh Bri2 BRICHOS R221E the primers 5′-CACCTGGGTTTCTTTATTTAT-GAACTGTGTCATGACAAGGAAAC-3′ and 5′-GTTTCCTTGTCATGACA-CAGTTCATAAATAAAGAAACCCAGGTG-3′ were synthesized. With the wildtype NT*-Bri2 BRICHOS (corresponding to the solubility tag NT* followed by Bri2 residues 113-231 26,41 ) plasmid as polymerase chain reaction (PCR) template Bri2 BRICHOS R221E (Addgene ID: 138134) was obtained with QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, US), and the DNA sequence was confirmed (GATC Bioteq, Germany). As described previously 26,41 , the proteins were expressed in SHuffle T7 competent Escherichia coli cells.…”

Section: Methodsmentioning

confidence: 99%

“…Rh Bri2 BRICHOS R221E was produced in fusion with a solubility tag, NT*, that was recently developed based on the Nterminal domain of spider silk proteins 26,41,42 . Purified NT*-Bri2 BRICHOS R221E was separated into oligomers, dimers, and monomers by size-exclusion chromatography (SEC; Fig.…”

Section: R221e Mutant Forms Stable Monomers and Unstable Oligomersmentioning

confidence: 99%

Augmentation of Bri2 molecular chaperone activity against amyloid-β reduces neurotoxicity in mouse hippocampus in vitro

et al. 2020

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Molecular chaperones play important roles in preventing protein misfolding and its potentially harmful consequences. Deterioration of molecular chaperone systems upon ageing are thought to underlie age-related neurodegenerative diseases, and augmenting their activities could have therapeutic potential. The dementia relevant domain BRICHOS from the Bri2 protein shows qualitatively different chaperone activities depending on quaternary structure, and assembly of monomers into high-molecular weight oligomers reduces the ability to prevent neurotoxicity induced by the Alzheimer-associated amyloid-β peptide 1-42 (Aβ42). Here we design a Bri2 BRICHOS mutant (R221E) that forms stable monomers and selectively blocks a main source of toxic species during Aβ42 aggregation. Wild type Bri2 BRICHOS oligomers are partly disassembled into monomers in the presence of the R221E mutant, which leads to potentiated ability to prevent Aβ42 toxicity to neuronal network activity. These results suggest that the activity of endogenous molecular chaperones may be modulated to enhance anti-Aβ42 neurotoxic effects.

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“…The pH‐sensitive N‐terminal of silk enables the silk protein to stay in solution at very high protein concentrations; thus, recombinant fusion of the N‐terminal domain converted an aggregation‐prone therapeutic protein to its hypersoluble counterpart (this technology is now marketed as SolvNT) . This strategy also simplified protein purification, improved yields, and allowed the expression of nontransmembrane proteins that are otherwise refractory to recombinant production.…”

Section: (Old) New Silk Industries: Opportunities and Challenges For mentioning

confidence: 99%

The Biomedical Use of Silk: Past, Present, Future

Holland

Numata

Rnjak‐Kovacina

et al. 2018

Adv Healthcare Materials

585

509

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Humans have long appreciated silk for its lustrous appeal and remarkable physical properties, yet as the mysteries of silk are unraveled, it becomes clear that this outstanding biopolymer is more than a high‐tech fiber. This progress report provides a critical but detailed insight into the biomedical use of silk. This journey begins with a historical perspective of silk and its uses, including the long‐standing desire to reverse engineer silk. Selected silk structure–function relationships are then examined to appreciate past and current silk challenges. From this, biocompatibility and biodegradation are reviewed with a specific focus of silk performance in humans. The current clinical uses of silk (e.g., sutures, surgical meshes, and fabrics) are discussed, as well as clinical trials (e.g., wound healing, tissue engineering) and emerging biomedical applications of silk across selected formats, such as silk solution, films, scaffolds, electrospun materials, hydrogels, and particles. The journey finishes with a look at the roadmap of next‐generation recombinant silks, especially the development pipeline of this new industry for clinical use.

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Efficient protein production inspired by how spiders make silk

Cited by 111 publications

References 80 publications

High-yield Production of Amyloid-β Peptide Enabled by a Customized Spider Silk Domain

High-yield Production of Amyloid-β Peptide Enabled by a Customized Spider Silk Domain

Augmentation of Bri2 molecular chaperone activity against amyloid-β reduces neurotoxicity in mouse hippocampus in vitro

The Biomedical Use of Silk: Past, Present, Future

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