Method for improving extraction of spider silk proteins

A spider silk protein and western blotting technology, applied in the field of improving the extraction of spider silk proteins, can solve the problems of low solid or fiber toughness, poor hand feeling, and poor yield, etc.

Pending Publication Date: 2022-03-22
BOLT THREADS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Solubilization of these proteins often requires harsh chemical conditions for biomolecules, which often degrades the protein, resulting in poor yields, and solids or fibers with low tenacity and poor hand

Method used

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  • Method for improving extraction of spider silk proteins
  • Method for improving extraction of spider silk proteins
  • Method for improving extraction of spider silk proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0159] Embodiment 1: Calcium salt extraction

[0160] Highly crystalline silk forms aggregates in solution, leading to reduced solubility and thus reduced recovery from host cells during production. Therefore, there is a need for improved methods of dissolving such crystalline silk. The method described in these examples is the use of calcium salts and alcohols to increase the solubility of silk proteins.

[0161] Materials and methods

[0162] The UDMisp64k protein (also referred to as P0 (representative block amino acid sequence shown in SEQ ID NO. 23)) was extracted using various calcium salts to identify optimal calcium salts. P0 is an exemplary highly crystalline silk protein. Escherichia coli was transformed with an expression vector containing a P0 silk gene fused to a 6x His tag (six histidines (GGGGG-HHHHHH) attached to the c-terminus of P0 with a glycine linker) in the presence of chloramphenicol. Broth (Terrific Broth, defined basal salt medium) growth. After 2...

Embodiment 2

[0166] Embodiment 2: alcohol extraction

[0167] Alcohol selection was investigated to determine optimal extraction conditions. First, mix insoluble P0 with CaCl 2 Incubate together in water or in methanol to determine if alcohol solvent needs to be included. Next, substitute ethanol and isopropanol as the main solvents. Finally, water was introduced as a solvent along with methanol to reduce the volatility of the process.

[0168] Materials and methods

[0169] P0 was expressed in E. coli cells as described in Example 1. Cells were lysed using a Microfluidic Nanohomogenizer, and insoluble material was pelleted via centrifugation. As shown in Table 4, CaCl in different solvents were prepared 2 solutions with different concentrations.

[0170]

[0171] 100 mg of insoluble cellular material was added to 1 ml of each solution and resuspended via pipetting. Samples in solution conditions 1-6 were incubated for 1 hour at room temperature (approximately 22°C). Parallel s...

Embodiment 3

[0181] Example 3: Incubation time and temperature

[0182] The extraction temperature was varied to determine the optimum temperature to achieve maximum extraction while minimizing extraction time. Agitation of the samples was also introduced. Lowering the temperature along with continuous mixing was investigated as a more scalable process option.

[0183] Materials and methods

[0184] P0 was expressed in E. coli cells as described in Example 1. Cells were lysed using a Microfluidic Nanohomogenizer, and insoluble material was pelleted via centrifugation. 1 ml of 2M CaCl in methanol 2 The solution was added to 100 mg of insoluble cell material, which was resuspended via pipetting. Twelve aliquots were prepared. Six aliquots were incubated with stirring at 35°C for 0, 15, 30, 60, 120 and 240 min. The remaining 6 aliquots were incubated with agitation at 55°C for 0, 5, 15, 30, 60 and 120 min. At each time point, samples were removed and centrifuged at 15,000 x g in a tab...

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Abstract

Provided herein are methods for improving the dissolution, extraction and isolation of recombinant spider silk proteins with salt and alcohol buffers. Provided herein are methods of solubilizing a recombinant spider silk protein from a host cell comprising: providing a cell culture comprising a host cell wherein the host cell expresses a recombinant spider silk protein; collecting an insoluble fraction derived from the cell culture, wherein the insoluble fraction comprises the recombinant spider silk protein; the insoluble portion of the host cell is added to a solution comprising a salt and an alcohol, thereby solubilizing the recombinant spider silk protein in the solution.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application No. 62 / 890,473, filed August 22, 2019, which is hereby incorporated by reference in its entirety. [0003] sequence listing [0004] This application contains a Sequence Listing, which was submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy was created on XX / XX / 20XX and is named XXXXXUS_sequencelisting.txt and is X,XXX,XXX bytes in size. Background technique [0005] Spider silk polypeptides are large (>150kDa, >1000 amino acids) polypeptides that can be broken down into three domains: N-terminal non-repeat domain (NTD), repeat domain (REP) and C-terminal non-repeat domain (CTD ). The NTD and CTD are relatively small (about 150, about 100 amino acids, respectively), are well studied and are believed to confer water stability, pH sensitivity and molecular arrangement upon aggregation to polypeptides. NTDs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/22C07K14/435C12N15/12C12P21/02D01F9/00
CPCC12P21/02D01F4/00C07K14/43518C07K2319/02C07K2319/21C07K1/12C07K1/34C12N15/70G01N33/6848D01F1/00
Inventor 梅嘉恒S·李R·B·穆塔利克C·R·彼得森
Owner BOLT THREADS
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