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WO2016116767A1 - Nouvelle technique d'extraction de protéines de soie - Google Patents

Nouvelle technique d'extraction de protéines de soie Download PDF

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Publication number
WO2016116767A1
WO2016116767A1 PCT/GB2016/050143 GB2016050143W WO2016116767A1 WO 2016116767 A1 WO2016116767 A1 WO 2016116767A1 GB 2016050143 W GB2016050143 W GB 2016050143W WO 2016116767 A1 WO2016116767 A1 WO 2016116767A1
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Prior art keywords
silk
arthropod
partially
denatured
glands
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Friedrich Wilhelm Ludwig Paul Vollrath
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • D01F4/02—Monocomponent artificial filaments or the like of proteins; Manufacture thereof from fibroin

Definitions

  • the invention relates to processes of obtaining silk glands from silk-producing arthropods (preferably silkworms) and to processes of preparing raw silk fibroin obtained directly from the silk glands of silk-producing arthropods.
  • the invention also relates to raw silk fibroin produced by such processes.
  • Silks are natural proteins which have been used as fibres for many thousands of years, e.g. in textiles. Most commonly, silk is obtained from cocoons of the mulberry silkworm Bombyx mori which was domesticated thousands of years ago. Other taxa of silkworms have great potential for commercial use of their silk proteins: these include silk-producing larvae of the bombycid, iasiocampid, satumid and thaumatopoid species.
  • saturnid species of wild silkworms which are currently used for commercial silk production include Antheraea yamamai, Antheraea pernyi, Antheraea mylitta, Antheraea assama and Philosamia cynthia spp.
  • Lasiocampid silkworms include, for example, Gonometa species that also have considerable potential for commercial silks. There are also many spiders with interesting and potentially important silks for commercial applications, with Nephila species being one of the most well-known.
  • Silk is spun by the animal (e.g. spider or silk worm) through a complex process that involves both mechanical shearing and chemical treatment. In all cases of natural spinning, the silk is dehydrated and denatured in controlled ways in order to transition the liquid raw silk dope into a solid filament, fibre or ribbon. This spinning transition leads to strong molecular interactions which are very difficult to break.
  • animal e.g. spider or silk worm
  • silks are usually commercially obtained either as fibres which are collected from spun cocoons (e.g. CN 281 1337 Y), by direct reeling from the animal (e.g. WO2012/080510) or by extracting fresh glands immediately from a recently-killed animal (e.g. WO03/037925).
  • Spun silks can be dissolved and turned into liquids called reconstituted silk fibroins or RSF. All silk reconstitution (i.e. RSF) production methods aim to reverse some of strong molecular interactions in the silk fibres so that the silks can be converted back into a liquid that can then be spun again or otherwise be converted into a solid material.
  • RSF silk reconstitution
  • biocompatible, implantable, substantially sericin-free silk fabric e.g. US 8,685,426 B2
  • cartilaginous medical implants e.g. WO2009/133532.
  • Such partial denaturing might mean a denaturing of the outside of the gland with the raw silk fibroins inside the gland being not denatured but encased in a coating of fully denatured or partially denatured silk.
  • such partial denaturing might also denote a partial denaturing of all silk proteins inside the gland.
  • Silk 1 Raw silk in the gland is often called Silk 1 while spun (i.e. denatured) silk is often called Silk 2 (see 'Fibroin' in www. wikipedia.org).
  • Silk 2 Raw silk in the gland is often called Silk 1 while spun (i.e. denatured) silk is often called Silk 2 (see 'Fibroin' in www. wikipedia.org).
  • a typical descriptor of the differences between the two conformations are perceived conversions of and ratios between alpha- helical and beta-sheet molecular conformations (Drummy ei a/. 2005, Thermally induced alpha-helix to beta-sheet transition in regenerated silk fibers and films. Biomacromolecules 6:3328-33, and references therein) as well as conversions from beta-turn/spiral to anti-paraliel pleated beta-sheets (Yamane et a/.
  • the invention allows for a wide range of manipulation of the denaturation conversion from Silk I to Silk II depending on the durations and strengths of the chemicals used for the preservation.
  • said preservation allows storage in situ in the equally-preserved worm for weeks perhaps months depending on the preservation conditions.
  • This aspect of the invention allows for the availability of silk glands independent of the availability of live worms and this liberates the subsequent processing from many time constraints as anyone skilled in the art will appreciate.
  • preservation allows for the production and storage of silk material that has not been exposed to the natural conditions associated with natural spinning; these typically include ambient and natural pollutants. For example, in many commercially-important wild silks the worm always voids gut contents onto the cocoon; these typically containing highly toxic compounds.
  • said preservation greatly facilitates the extraction of the preservation- hardened gland from the worm and its removal of the surrounding animal and connective tissue.
  • extracting a fresh gland from a silkworm or spider is difficult and time consuming work with a significant chance that the silk in the gland will congeal in the process of extraction.
  • the removal of the tissue and cells surrounding the gland is also very difficult in fresh glands
  • said preservation allows for easy removal of all of the sericin proteins by gentle washing off the sericin-gum coating that covers the outside of parts of the gland.
  • said preservation allows the harvesting of glands that, after removal from the worm, can be more easily efficiently and economically further processed than silk taken from spun cocoons.
  • processing might include lyophilizing, freeze drying for storage as solid or powder, or storing directly in buffered osmolytes such as honey which is traditionally used for meat stabilisation and is effective for general protein stabilisation (Wong et a/. 2013 Honey-Induced Protein Stabilization as Studied by Fluorescein isothiocyanate Fluorescence, The Scientific World Journal, Vol 2013 Article ID 981902, 8 pages, http://dx.doi.org/10.1 155/2013/981902).
  • the silk glands whether stored whole or cut-up, are then either directly or after intermediate storage dissolved in one or several of the various chaotropes commonly used on silk filaments to isolate and extract the silk protein molecules.
  • the said preserved glands are only partially denatured and the constituent silk molecules have not passed through the natural spinning and post-spinning denaturing and tanning processes; this allows for less harsh conditions to effect the harvesting of the silk proteins as anyone skilled in the art will appreciate.
  • said preservation prevents the silk molecules from the full dehydration and denaturation associated with the natural spinning process it allows the harvesting of silk protein molecules that are closer to the unspun than to the spun state. This has implications for the production of high quality RSF that is similar to natural unspun silk as it prevents the alpha to beta transitions commonly associated with the phase transitions associated with natural silk spinning. As will be shown herein, comparative measurements can be taken both on silks reconstituted from spun cocoons and reconstituted from preserved glands.
  • said preservation allows for the harvesting of silk not only from the domesticated mulberry silkworm, but also from wild silks which are much more difficult to degum and demineralise and moreover also much more difficult to dissolve in chaotropic agents such LiBr.
  • the required washing and other treatments provide conditions that facilitate molecular cross-linking which in turn require further treatments that themselves are damaging to the molecular integrity of the long-chained silk proteins.
  • preserving glands inside the animal provides many advantages for silk molecule extraction over extraction from spun and stored silk cocoons and also over extraction of glands from fresh worms. From the start, the silk material obtained from preserved worms and glands has not been exposed to pollutants typically associated with natural cocoon building. Preservation provides a less labour intensive and energetically more
  • Extracting the intact silk gland from a silkworm or spider according to the invention is exceptionally easy and straightforward, and indeed much easier and simpler than dissecting silk glands from untreated animals.
  • the glands thus preserved are of a rubbery nature as is apparent in Fig. 1 and they are easily pulled free of the worm.
  • the sericin coating is then easily washed off.
  • the silk fibroin molecules are partially denatured either throughout the gland or only on the surface of the gland which would protect the native raw silk inside the lumen of the gland.
  • the production of near-natural silk protein molecules by processes of the invention also circumvents the need to use fresh silk extracted from newly-killed silkworms as disclosed in WO03/037925.
  • the current invention may significantly increase the speed of extraction of the silk glands compared with extraction from freshly-killed silkworms or with obtaining silk from cocoons.
  • the invention provides a process for obtaining the silk glands from a silk-producing arthropod, the process comprising the steps:
  • the invention also provides a process for preparing raw silk fibroin from the silk glands of a silk-producing arthropod, the process comprising the steps:
  • the invention also provides a partially-denatured raw silk fibroin which is obtained or obtainable by a process of the invention.
  • the silk-producing arthropod is preferably a silk-producing insect, arachnid or crustacean.
  • the silk-producing insect is a silkworm.
  • the silkworm is a mulberry silkworm (e.g. the domesticated Mulberry Silkworm, Bombyx mori) or non-mulberry silkworm also often called wild (non- domesticated) silkworm.
  • mulberry silkworm is used herein for the larvae of the Lepidopteran Bombyx mori and the term "wild silkworm” is used for the larvae of moths of the families Bombycoidea (other than B. mori), Saturnidae, Lasiocanpidae, Mimaiionidae,
  • non-mulberry silk worms or wild (non-domesticated) silkworms include Antheraea ssp., for example, Antheraea assama, Antheraea miiitta, Antheraea pernyi, Antheraea yamamai and Philosamia ssp. for example, Philosamia Cynthia ricini and Philosamia Cynthia pryeri.
  • Saturnid worms may also be used, such as those of the genus Actias or Cecropia. Although these are not generally defined as wild silk worms, they yield a closely-similar silk and can be used in this invention.
  • the silk worm is the domesticated Mulberry Silkworm (Bombyx mori).
  • the silk-producing arthropod is a silk-producing spider.
  • spiders examples include spiders of the Nephilidae, Araneidae, Latrodectidae and Pisaurida families.
  • the silk-producing arthropod is a silk-producing crustacean, e.g. an amphipod crustacean such as the silk 'shrimp' Crassicorophium boneiiii and their relatives which produce underwater salt tolerant silks (Kronenberger et ai. 201 1 A novel marine silk. Naturwissenschaften, 99: 3-10).
  • a silk-producing crustacean e.g. an amphipod crustacean such as the silk 'shrimp' Crassicorophium boneiiii and their relatives which produce underwater salt tolerant silks (Kronenberger et ai. 201 1 A novel marine silk. Naturwissenschaften, 99: 3-10).
  • the silk glands are the glands within the silk-producing arthropod that produce raw silk dope. During the partial denaturing step of the invention, the silk glands will still be located within the body of the silk-producing arthropod.
  • the glands of the silkworm comprise an epithelial layer of tissue consisting largely of a layer of columnar cells surrounded by a basement membrane of structural proteins. Inside the epithelium, in the anterior part of the gland, there is a coating of sericin made up of five or so different sericin layers. The coating of sericin surrounds a thick core of fibroin.
  • in situ means that the raw silk fibroin is partially-denatured in the silk glands themselves whilst the silk glands are still in the silk-producing arthropod.
  • the silk fibroin is partially-denatured in the silk glands before the silk glands are removed from the silk-producing arthropod.
  • the raw silk fibroin in the silk glands is partially denatured whilst the silk-producing arthropod is whole or intact, i.e. the raw silk fibroin in the silk glands is partially denatured before the silk-producing organism has had any of its tissues or organs removed. ln some embodiments, one or more tissues or organs are removed from the silk- producing arthropod before the raw silk fibroin in the silk glands is partially denatured. For example, the silk-producing arthropod may be decapitated before the raw silk fibroin in the silk glands is partially denatured.
  • part of the silk gland is removed before the raw silk fibroin in the silk glands is partially denatured.
  • the raw silk fibroin in the silk glands is partially denatured whilst the silk glands are intact or essentially intact, i.e. before some or all of the silk glands are removed.
  • raw silk fibroin also referred to herein as silk dope
  • silk dope refers to the silk protein which may be extracted from the silk glands of the silk-producing arthropod.
  • the extracted silk protein solution generally comprises both sericin and fibroin proteins.
  • partially-denaturing raw silk fibroin means that the raw silk fibroin is treated in such a way so as to increase the denaturation state of the raw silk fibroin.
  • the denaturation state of the raw silk fibroin is increased (depending on requirements significantly increased) relative to the non-denatured state of raw silk fibroin from control silk glands which have been directly obtained from a freshly-killed arthropod of the same species.
  • the ratio of alpha-helix to beta-sheet conformation of the raw silk fibroin is changed with the latter increased (preferably significantly increased) measurably relative to the degree of beta-sheet conformation of raw silk fibroin from control silk glands which have been directly obtained from a freshly-killed arthropod of the same species.
  • the raw silk fibroin is not completely denatured. Denaiuration of the raw silk fibroin may be quantified by any suitable technique, e.g. a thermal shift assay, also called Differential Scanning Fluorimetry (DSF).
  • DSF is a therma!-denaturation assay that measures the thermal stability of a target protein.
  • the DSF is performed in
  • a further method of determining the thermal stability change is to obtain a thermal denaturation curve by fluorescence spectroscopy using a fluorescent dye, such as Sypro® Orange (Sigma Aldrich).
  • Sypro® Orange binds non-specificaiiy to
  • fluorescence-labelled antibodies may be used, for example, anti-fibroin antibodies labelled with green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • the stability curve and its midpoint value (melting temperature, T m ) are obtained by gradually increasing the temperature to unfold the protein and measuring the fluorescence at each point. Curves may compare two proteins, and AT m may be calculated.
  • a fluorescence-based thermal shift assay can be performed on instruments that combine sample temperature control and dye fluorescence detection, such as real-time polymerase chain reaction (RT-PCR) machines.
  • RT-PCR real-time polymerase chain reaction
  • the difference (AT m ) in the T m between the partially-denatured raw silk fibroin produced in accordance with the invention and raw silk fibroin from control silk glands which have been directly-obtained from a freshly-killed arthropod of the same species (i.e. which have not been partially denatured) is at least 1 2, 3, 4, 5, 6, 7, 8, 9 or 10°C.
  • the range of expected values may also be taken for the correlation curve of FSD/FTIR v. reversing heat capacity DSC plot (e.g. as in Xiao Hu, David Kaplan and Peggy Cebe. Determining Beta-Sheet Crystaiiinity in Fibrous Proteins by Thermal Analysis and infrared Spectroscopy. Macromoiecuies 2008, 39, 8161 -6170).
  • Tj (Voiirath et a/. 2014) instead of T m of the silk fibroins.
  • T denotes the temperature- induced instability of hydrogen interactions. Shortly after reaching the instability temperature, Tj, the protein rapidly aggregates causes dye dissociation and an associated decrease in fluorescence.
  • the first derivative of the peak intensity profile highlights these key transitions, in the same way as DSC.
  • a graph of the first derivative of the fluorescence intensity against temperature of the partially-denatured silk fibroin solution of the invention in a DSF assay will show a significant decrease in the T, peak compared to the Tj peak obtained from a control silk fibroin solution which has been directly obtained from the silk glands of a freshly-killed silk-producing arthropod (e.g. Bombyx rnori).
  • a freshly-killed silk-producing arthropod e.g. Bombyx rnori
  • a graph of the first derivative of the fluorescence intensity against temperature of the partially-denatured silk fibroin solution of the invention in a DSF assay will show a significant increase in the Tj peak compared to the Tj peak obtained from a control RSF solution (e.g. from Bombyx rnori).
  • test and control silk protein solutions are analysed at an appropriate concentration.
  • the control silk protein solution should be of the same protein
  • concentration as the solution with which it is being compared.
  • appropriate concentrations include 0.5%, 1.0% and 1.5% (weight/volume) silk solutions in pure water.
  • RSF may be made using the industry benchmark standard as given in Rockwood et al. (201 1 , Nature Protocols 6, 1612-1630). Control RSF solutions will have been extracted with LiBr solution in order to remove sericin. Silk solutions of the invention will have been extracted with water in order to remove sericin prior to testing and analysis. The same fluorophore should be used in both test and control protein solutions.
  • control solution should be taken from the same part of the silk gland as the test solution, e.g. both from posterior-middle section or both from the posterior sections of the silk glands. (In silk worms, these sections are preferred in order to minimise the presence of sericin.)
  • the silk is preferably taken from the Major
  • the term "significant" may indicate at least a 20%, 30%, 40% or 50% increase/decrease in the T, peak height or integrated T, peak intensity.
  • the term "significant” may indicate a 0-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100% or 100-200% or 200-500% increase/decrease in the Tj peak height or integrated T, peak intensity.
  • the T, peak will be expected at about 67°C (1.0% w/v solution).
  • the T, peak will be expected at about 60°C (1 .14% w/v solution).
  • the Tj peak will be expected at about 48°C (1 .0% w/v solution).
  • the term "significant" may also refer to p ⁇ 0.05 (two tailed test).
  • the partial denaturation may be defined in terms of a partial or essentially-complete formation of the silk II conformation, characterised by the formation of ⁇ -sheet conformation.
  • the ⁇ -sheet conformation may be measured by measuring the glass transition temperature.
  • the degree of p-sheet conformation may be derived from the reversing heat capacity at the glass transition temperature, as measured, for example, by temperature-modulated differential scanning calorimetry.
  • the partially-denatured silk fibroin of the invention preferably has 2-5 fold greater
  • beta-sheet content as defined by modulated DSC/FSD FTIR than the beta-sheet content of a control silk fibroin solution which has been directly obtained from the silk glands of a freshly-killed silk-producing arthropod (e.g. Bombyx mori).
  • the partial denaturing may be performed by any suitable technique.
  • suitable techniques include: dehydrating the arthropod; drying the arthropod; freezing the arthropod; freeze-drying the arthropod; heating the arthropod; chemically-treating the arthropod; and fixing the arthropod.
  • the arthropod is frozen to a temperature of at least -5°C, -10°C or - 20°C.
  • the speed of freezing may influence the extent of the beta-sheet formation, with less beta-sheet being obtained if freezing is performed very rapidly, because speed reduces time for phase separation of the protein from the ice-rich phase, in some embodiments, the freezing may be performed in air at -4 °C; in ice/salt mixtures; or spray-drying or freezing in a very cold alcohol bath.
  • the temperature of the arthropod may then be raised again to room temperature, in other embodiments, the silk glands are removed from the arthropod whilst it is still frozen or partially-frozen.
  • the arthropod may be dried, dehydrated, freeze-dried or lyophilised.
  • the partial denaturation is produced by heating the arthropod.
  • the arthropod is heated to a temperature of at least 30°C to 35°C, 35°C to 40°C, or 40°C to 45°C.
  • the heating step will be of a short duration, e.g. 1 -5, 5-10, 10-20 or 20-30 seconds, or for 1 -2 or 2-5 minutes.
  • the heating may be carried out in a liquid, preferably an aqueous liquid, most preferably water.
  • the arthropod is chemically-treated to partially denature the raw silk fibroin in the silk glands.
  • chemical treating include treating the arthropod with an alcohol and/or an acid and/or brine or by submerging in alcohol or brine.
  • suitable alcohols include C2-C4 alcohols, e.g. ethanol and
  • isopropanol examples include organic acids, e.g. acetic acid, citric acid or acetate buffer between pH 4.5 to 6.8.
  • suitable solutions include 5-12% aqueous solutions (e.g. about 10% acetic acid), in some embodiments, vinegar may be used.
  • suitable brines include a solution of salt such as sodium chloride (NaCI) in water at concentrations ranging from 3% to 30%.
  • salting out This may be done with a reagent selected from the cation and anion Hofmeister series.
  • the chemical treatment may, for example, be for 1-5 minutes, 5-10 minutes, 10-
  • the arthropod is fixed with a fixative to partially denature the raw silk fibroin in the silk glands.
  • fixatives include ethanol and isopropanol.
  • the silk glands may extracted from the arthropod either by manual dissection or by mechanical methods, e.g. by ball-milling or freeze fracturing.
  • the extracted silk glands may then be washed to clean off unwanted tissues and/or unwanted coatings such as sericin or other compounds often found associated with natural silks. Washing may be done in pure water or Ringer Solution, or in other solvents, such as an alcohol.
  • the silk glands may be treated with one or more enzymes, e.g. trypsin.
  • the silk glands may be shredded and/or minced, e.g. by cutting up or by pounding or by sonication.
  • the silk glands may be stored for further processing at a later stage or processed straightaway.
  • the silk glands or the contents of the silk glands are placed in a solvent to dissolve the silk fibroin.
  • the extracted silk fibroin solution may be treated with a chaotropic agent such as lithium bromide or formic acid.
  • a chaotropic agent such as lithium bromide or formic acid.
  • the extracted silk protein solution may be treated with water or an aqueous solution to remove sericin or other easily-soluble compounds.
  • the extracted silk fibroin solution may be dialysed either before or after the chaotropic treatment.
  • the protein solution may then be stored, e.g. in a siliconized container, to prevent degradation during storage, preferably under silicone oil.
  • the protein solution can be used directly or can be diluted with water or other solutions or solvents before use or mixed with other components of the final product including pharmacologically active compounds by way of example only. It can be further treated. Prolonged storage is possible after freeze-drying the protein solution before or after dilution.
  • Examples of further treatment steps after extraction of the protein solution include treatment with aqueous or non-aqueous solutions or buffers or treatment with vapours from volatile acids or bases or volatile buffers.
  • a further example is the addition of mono-vending or divalent ions to condition the protein solution (e.g. to facilitate its conversion to a formed object for example by extrusion).
  • These conditioning solutions may be added to the protein solution through a semipermeable or porous membrane as described, for example, in WO 01/38614.
  • the protein solution may be treated by the addition of an aldehyde solution, e.g. giutaraldehyde, to cross-link the constituents of the protein solution.
  • an aldehyde solution e.g. giutaraldehyde
  • Other aldehydes or cross-linking agents could be used as will be understood by persons skilled in the art.
  • Other conditioning agents may be added to the protein solution.
  • the final processing of the protein solution can be achieved in a variety of ways.
  • the protein solution may be allowed to flow into a mould (e.g. WO2009/133532).
  • the solution is then treated with an aldehyde or other cross-linking agent in solution or as vapour applied directly or through a semipermeable membrane forming substantially all or part of the walls of the mould.
  • a similar aldehyde can be applied immediately before the spinning solution enters a blow moulding mould or an annular die used for blown extrusion.
  • the invention provides a partially-denatured silk fibroin which is obtained or obtainable by a process of the invention.
  • the silk fibroin may be in the form of solution, gel or solid.
  • the invention further provides a non-living silk-producing arthropod whose silk glands comprise partially-denatured raw silk fibroin.
  • the arthropod is a silk worm.
  • the arthropod is whole/intact or headless.
  • the invention further provides isolated silk glands from a silk-producing arthropod, wherein the raw silk fibroin in the silk glands is partially denatured.
  • the invention further provides a composition comprising a partially-denatured raw siik fibroin solution which is obtained or obtainable from a si!k-producing arthropod.
  • the difference (AT m ) in the T m is at least 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10°C.
  • the invention also provides a partially-denatured silk fibroin solution which is obtained or obtainable from a silk-producing arthropod, wherein the first derivative of the fluorescence intensity against temperature of the partially-denatured siik fibroin solution in a DSF assay shows a significant decrease in the T, peak compared to the peak obtained from a control siik fibroin solution which has been directly-obtained from the siik glands of a freshly-killed control silk-producing arthropod of the same species.
  • the invention further provides a partially-denatured siik fibroin solution which is obtained or obtainable from a silk-producing arthropod, wherein the first derivative of the fluorescence intensity against temperature of the partially-denatured siik fibroin solution in a DSF assay shows a significant increase in the T, peak compared to the T, peak obtained from a control RSF solution.
  • the invention also provides a partially-denatured silk fibroin solution which is obtained or obtainable from a silk-producing arthropod, wherein the partially-denatured silk fibroin solution has 2-5 fold greater (more preferably 5-10 fold or 10-20 fold greater) beta-sheet content as defined by modulated DSC /FSD FTIR than the beta-sheet content of a control siik fibroin solution which has been directly-obtained from the silk glands of a freshly-killed silk-producing arthropod.
  • the silk-producing arthropod is a silkworm or a spider, preferably wherein the silkworm is Bombyx mori.
  • the partially-denatured silk fibroin of the invention can be used in numerous applications.
  • a medical device e.g. a cartilage patch, or to provide the coatings of a nerve guide.
  • the medical device is an implantable medical device.
  • the medical device may, for example, be an arteriovenous (AV) graft for haemodialysis, a vascular graft, a bifurcation graft or an anastomosis device
  • AV arteriovenous
  • the invention further provides the use of a partially-denatured raw silk fibroin solution as disclosed herein in producing a medical device, preferably an implantable medical device.
  • FIG 1 Dissection of a Bombyx mori silkworm showing the siik glands hardened by partial denaturation preservation. Also visible is the tail (where much of the fibroin is secreted into the lumen) and the duct where the siik exits the gland into the spinning press situated in the head of the animal.
  • the central part of the gland may the preferred part for fibroin extraction, and the sericin coating (readily visible as milky white at the taper leading to the duct) can either be washed off or cut off and discarded together with the fibroin material in that section.
  • Figure 2 Overlay of DSF Protein preparation with Sypro® dye response traces of different Bombyx mori silk preparations (for details on the technique see Volirath et al.
  • Example 1 Fixation of silkworms in acetic acid
  • the instar was approximately 7cm long and contained a gland having several milligrams containing a high concentration of fibroin.
  • the protein dope contained fibroin coated with sericin.
  • the silkworm and silk gland within were preserved in 10% acetic acid for 2 weeks before the glands were obtained simply by cutting along the back of the worm and pulling the glands out.
  • the glands were washed gently for 2 hours in de-ionised water, and then cut up into small slices to extract the partially denatured silk.
  • Standard LiBr protocol was used to prepare the control RSF solution from degummed cocoon silk, i.e. dissolving in LiBr followed by dialysis before measuring the resultant solution by DSF.
  • a graph of the first derivative of the fluorescence intensity against temperature of the partially-denatured silk fibroin solution of the invention in a DSF assay showed a significant increase in the T, peak compared to the T, peak obtained from a control RSF solution (from Bornbyx mori). It is noted that the standard RSF shows no specific peaks indicating protein transition folding; the slope of the curve is attributed to signal drift.
  • the native silk fibroin which was directly extracted from the gland shows a distinct peak at 88°C while the invention-extracted Silk Fibroin shows two transition peaks.
  • different preservation denaturation conditions result in different DSF response curves which in turn affect the response of the materials for further processing.
  • a final instar of the silkworm, Bombyx mori, is selected as in Example 1.
  • the silkworm is killed and frozen or fully submerged in an ethanol alcohol:water solution.
  • the silk glands are removed by dissecting the silkworm.
  • the silk gland is then immersed in distilled water for two minutes.
  • the epithelium of the gland is removed using 1 hour maceration in an enzyme such as trypsin.
  • the gland is then cut-up in a blender and the protein dope mixture is immersed in LiBr.
  • the protein mixture is fixed into desired shape by administering a 2% giutaraldehyde solution in a 0.1 M phosphate buffer having a pH of 7.4 for twelve hours.
  • Example 3 Heat-treatment of silk worms
  • a final instar of the silkworm, Bornbyx mori, is selected as in Example 1.
  • the silkworm is killed and fixed in 20% brine solution to partially-denature the proteins and to preserve them in that stage.
  • the silk glands are removed as described in Example 1 and the epithelium removed.
  • the protein mixture is placed into distilled water and agitated for around 30 minutes.
  • the sericin protein is dissolved into the distilled water to leave substantially fibroin protein in the protein mixture.
  • the fibroin protein is blotted dry and placed into a mould for storage.

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Abstract

L'invention concerne des procédés d'obtention de glandes séricigènes d'arthropodes producteurs de soie (de préférence les vers à soie ) et des procédés de préparation de fibroïne de soie brute obtenue directement à partir des glandes séricigènes d'arthropodes producteurs de soie. L'invention concerne également la fibroïne de soie brute produite par de tels procédés.
PCT/GB2016/050143 2015-01-23 2016-01-22 Nouvelle technique d'extraction de protéines de soie Ceased WO2016116767A1 (fr)

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GBGB1501134.9A GB201501134D0 (en) 2015-01-23 2015-01-23 Raw material for bio-spinning casting
GB1501134.9 2015-01-23

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CN109954003A (zh) * 2019-05-05 2019-07-02 北京颢美细胞基因生物技术有限公司 沙蚕精液胞浆素及其制备方法与应用
CN116334923A (zh) * 2023-03-27 2023-06-27 中国科学技术大学 一种丝织品的加固方法、装置、设备及存储介质
CN116660216A (zh) * 2023-04-03 2023-08-29 浙江理工大学 基于多基团修饰磁珠和荧光量子点检测古代丝织品的方法

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Publication number Priority date Publication date Assignee Title
CN109954003A (zh) * 2019-05-05 2019-07-02 北京颢美细胞基因生物技术有限公司 沙蚕精液胞浆素及其制备方法与应用
CN109954003B (zh) * 2019-05-05 2022-08-16 北京颢美细胞基因生物技术有限公司 沙蚕精液胞浆素及其制备方法与应用
CN116334923A (zh) * 2023-03-27 2023-06-27 中国科学技术大学 一种丝织品的加固方法、装置、设备及存储介质
CN116660216A (zh) * 2023-04-03 2023-08-29 浙江理工大学 基于多基团修饰磁珠和荧光量子点检测古代丝织品的方法

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