Engineered spider silk proteins and uses thereof

A spider silk protein and sequence technology, applied in biochemical equipment and methods, peptide sources, peptides, etc., can solve problems such as undetermined effects

Active Publication Date: 2019-04-26
SPIBER TECHNOLOGIES AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The effect of pH on CT has not been determined and different effects have been observed

Method used

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  • Engineered spider silk proteins and uses thereof
  • Engineered spider silk proteins and uses thereof
  • Engineered spider silk proteins and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0254] Example 1: Generation of chimeric miniature spidroin NT2RepCT

[0255] The present inventors designed a miniature spidroin protein comprising NT from E. australis MaSpl and CT from E. australis MiSp bracketing a short repeat region from E. australis. The chimeric NT2RepCT protein was produced at unprecedentedly high levels in shake flask E. coli cultures and the yield after purification was about 125 mg protein / L cell culture. Almost all proteins are soluble after expression and cleavage, and bind efficiently to Ni-NTA columns ( figure 2 A). The eluate contained >95% pure NT2RepCT, and the protein size on the SDS PAGE gel corresponded well to the expected molecular mass (33 kDa) ( figure 2 A). Size exclusion chromatography indicated a mass of 100 kDa ( Image 6 ), in good agreement with the dimer (due to the constitutive dimeric nature of CT) and the non-globular structure of the repeat.

Embodiment 2

[0256] Example 2: Chimeric miniature spidroin NT2RepCT exhibits extreme solubility

[0257] Obtaining high concentration of spinning dopes in water has been a long-term main goal, but to date, even though non-physiological solvents have been used, the concentration of artificial spinning dopes has been reported to be in the range of 10%-30%. In terms of solubility, the NT2RepCT of Example 1 far exceeds all expectations; it can be concentrated to 500 mg / ml in an aqueous buffer at pH 8 without precipitation out, a concentration equal to or even greater than that found in spider spinning dope protein concentration. At such high concentrations, the protein forms a yellow hydrogel ( figure 2 B). Natural spider silk stocks have been shown to store as micelles, 100nm-200nm in diameter, likely with terminal domains in the shell and repeat regions occluded in the core. This has also been proposed as a storage mechanism in silk glands. The NT2RepCT protein behaves like native silk ...

Embodiment 3

[0259] Example 3: Biomimetic spinning of NT2RepCT miniature spidroin

[0260] Another prerequisite that needs to be fulfilled in order to realize biomimetic spinning is to design a spinning device that can mimic the conditions of the spider silk gland. We designed a first-generation simple yet effective spinning device from a thin, drawn glass capillary through which a highly concentrated NT2RepCT stock solution was pumped into a collection bath of acidic aqueous buffer ( image 3 ). This setting causes a drop in pH and allows shear forces to act on the stock solution as it passes through the tip of the capillary and leads to the formation of continuous solid fibers ( image 3 A- image 3 B). Fibers can be easily wound in air onto a rotating frame in lengths exceeding hundreds of meters ( image 3 C). Fibers can be spun into dope concentrations ranging from 100 mg / ml to 500 mg / ml. Fibers spun from dopes with concentrations >200 mg / ml are easier to handle and can be spun ...

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Abstract

A recombinant spider silk protein, consisting of no more than 800 amino acids, comprising a set of domains arranged according to the formula (NT)-REP-CT, wherein: the optional NT-domain, if present, comprises a sequence of 100 to 160 amino-acid residues derived from the N-terminal domain of a spider silk protein; the REP-domain comprises a sequence of 30 to 600 amino acid residues derived from therepetitive segment of a spider silk protein; and the CT-domain comprises a sequence of 70 to 120 amino acid residues derived from the C-terminal domain of a spider silk protein selected from: a sequence of 72 to 110 amino acid residues derived from the C-terminal domain of a spider silk protein, wherein the sequence comprises at least 7 residues independently selected from K, R, E and D; a sequence having at least 85% identity to SEQ ID NO: 15 or any one of SEQ ID NO:s 62-65 or 67-73; and a sequence having at least 70% identity to SEQ. ID NO: 64 or any one of SEQ ID NO:s 62-65 or 67-73, wherein the sequence comprises at least 7 residues independently selected from K, R, E and D. Solutions, polymers, uses and methods of manufacture thereof.

Description

technical field [0001] The present invention relates to the field of engineered spider silk proteins, and methods for producing fibers of such proteins. Background of the invention [0002] Spider silk consists of spidroin, which is produced in the abdominal glands. Most spiders produce as many as seven different types of silk in different glands that serve specific purposes and have different mechanical properties. The two toughest types of silk produced by orbweavers include the tractor silk (from the large ampullate gland) and the small ampullate silk (from the small ampullate gland). Most spidroin proteins, including the major ampullate (MaSp) and the small ampullate (MiSp), share a non-repetitive N-terminal domain (NT), an extensive repeat region (REP), and a non-repetitive C-terminal structure Common structure of the domain (CT). Since spiders are endemic and produce small amounts of silk, any industrial application of spider silk requires the production of recombin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435
CPCC07K14/43518B33Y70/00C12N5/0018C12N5/0062C12N2513/00C12N2533/50
Inventor 安娜·里辛扬·约翰松马琳·安德森
Owner SPIBER TECHNOLOGIES AB
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