EP2389387A2 - Natural biodegradable adhesive from the silk - Google Patents

Natural biodegradable adhesive from the silk

Info

Publication number
EP2389387A2
EP2389387A2 EP10747069A EP10747069A EP2389387A2 EP 2389387 A2 EP2389387 A2 EP 2389387A2 EP 10747069 A EP10747069 A EP 10747069A EP 10747069 A EP10747069 A EP 10747069A EP 2389387 A2 EP2389387 A2 EP 2389387A2
Authority
EP
European Patent Office
Prior art keywords
silk
sericin
seric
glues
adhesive
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10747069A
Other languages
German (de)
English (en)
French (fr)
Inventor
Barbara Kludkiewicz
Frantisek Sehnal
Michal Sery
Michal Zurovec
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biologicke Centrum AV CR V V I Ustav Molekularni Biologie Rostlin
Original Assignee
Biologicke Centrum AV CR V V I Ustav Molekularni Biologie Rostlin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biologicke Centrum AV CR V V I Ustav Molekularni Biologie Rostlin filed Critical Biologicke Centrum AV CR V V I Ustav Molekularni Biologie Rostlin
Publication of EP2389387A2 publication Critical patent/EP2389387A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • 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

Definitions

  • the present invention is based on the identification of the silk protein sericin 2 A that has glue properties. Natural sericin 2A and recombinant proteins of identical or similar amino acid sequences are proposed for the use as adhesives.
  • Natural adhesives have been used by various human cultures since ancient times and their efficiency may be verified after millennia.
  • the best known glue of animal origin is the bone glue (known from the Egyptian tombs and shown to be used already by ancient Sumerians), which is prepared by prolonged boiling of animal bones, skin, or hooves.
  • Another commonly used adhesive, the casein, which is made from milk precipitated in alkaline water, is more resistant to moisture and aging than the bone glue.
  • the best known adhesives of plant origin include starch, cellulose and rubber.
  • the first patent concerning an adhesive was issued in Germany around 1750 for the fish glue. Since then, a number of other patents were registered on natural rubber, starch, milk casein, and other products. Starch processing yielding an adhesive comparable to the bone glue has been known since 1896.
  • Starch is at present one of the most commonly used glues and it is usually produced from corn, tapioca, saga, wheat or potatoes. Natural glues are also obtained from some bacteria. Disadvantages of commonly used natural glues include their relatively low strength and low moisture resistance. The only exception is represented by the adhesive plaque protein secreted by the sea mussel Mytilus edulis, which has high tensile strength and hardens under water or on wet surfaces.
  • Caterpillars of a number of moths notably of the silkworm, Bombyx inori, pupate in cocoons that are spun from the silk fibers.
  • the silk is produced in a pair of silk glands and secreted as two filaments made of fibroin proteins; the filaments are enveloped and glued together into a single fiber by the proteins called sericins.
  • sericins There are several (6-9) sericin types present in the silk fiber coating.
  • the technology of raw silk manufacture involves dipping cocoons in hot and slightly alkaline water that dissolves the external sericin layer. Fibers loosened from several cocoons are apposed in the weaving machine and residual sericins on their surface glue them into a single thread. Pulling wet thread through an orifice controls its diameter.
  • sericins are adhesive and differ by their solubility in water.
  • sericins with highest adhesiveness are present in the silk glands from day 3 to day 5 of the last larval instar.
  • Two highly adhesive proteins accumulate in the anterior third of the middle silk gland region at that time
  • the highly adhesive silk protein was identified and structural requirements needed for its glue properties were analyzed. It is proposed to use this protein and its structurally defined derivatives as biodegradable glues. Using biochemical and molecular methods the applicants isolated the silkworm gene sericin 2 and demonstrated that it encodes two large proteins of 230 kDa and 120 kDa, which accumulate in the silk glands on days 3-5 of the last larval instar as a highly adhesive material. The production of two proteins, which were named Ser2A (composed of 1740 amino acid residues) and Ser2B (882 residues) is due to alternative splicing of the primary gene transcript. The mRNA encoding Ser2A includes all exons, while the mRNA for Ser2B lacks the largest 9 th exon.
  • Sericin or sericin-like glues can be prepared in 4 different ways: protein extraction from the silkworm silk glands; gentle sericin extraction from the cocoons; expression of natural or synthetic sericin 2 gene in suitable vector, e.g. bacteria or yeast; expression of artificial genes that encode combinations of the SERIC motifs.
  • the first method was used (Example 1) to prepare sericin 2 for the tests of its adhesive properties.
  • the second method was attempted but the preparations exhibited low tenacity; on the other hand, however, the stickiness of silk fibers obtained from the cocoons indicated that suitable technology could be developed.
  • Recombinant sericin-like adhesive proteins could be derived from cloned exon 9 of the Ser 2 gene but we preferred to use a smaller synthetic gene containing variations of the SERIC motif (Example 3).
  • Another synthetic gene was derived from a combination of the SERIC 1 and SERIC 2 motifs (Example 4).
  • Glues based on sericin harden in watery environment can be therefore used in many applications where the synthetic glues fail. Since silk threads containing surface sericins have been used in medicine as sutures, they do not induce immunogenic response. Sericin-based glues are therefore suitable for applications on the body surface (fixation of dentures, plasters for wound cover) and also internally (assembly of broken bones).
  • the sericin glue was distinctly better than the commercial starch glue but was inferior to the bone glue (Fig. 6). It is necessary to take into account, however, that very crude preparations of the sericin-type glues were compared with commercial glues that had been optimized for more than hundred years. Also, both the bone and starch glues harden only when the water evaporates, whereas sericin glues can harden under water.
  • Glues soluble in water and biodegradable are more environment-friendly than the synthetic glues that must be dissolved in organic solvents.
  • hardening of the currently used natural glues is often slow and depends on the evaporation of water.
  • Sericin glues have a great advantage in the rapidity of their hardening. Since silk is not immunogenic or allergenic (silk threads were used in surgery until synthetic threads were developed) the sericin glues are applicable in medicine.
  • Fig. 1 Amino acid sequence of the EX9 peptide that is encoded by exon 9 of the Ser2 gene (sequence No. 1). The peptide contains 44 repeats of the pentadecapeptide SERIC.
  • Fig. 2 Comparison of amino acid sequences of a portion of EX9 peptide, abyssal glue of Mytilus edulis (sequence taken from Filipula, D.R.; Lee, S.M.; Link, R.P.; Strausberg, S. L.; Strausberg, R.L., Biotechnol. Prog. 1990, 6, 171-177), and adhesive protein of trypanosoma (sequence from Chuenkova, M.; Pereira, M. E. J Exp. Med. 1995, 181, 1693-1703). Amino acids are numbered from the amino-terminus.
  • Fig. 3 Amino acid sequence of motif SERIC 1 that is derived from the repeats in the EX9 peptide (cf. Fig. 1).
  • Fig. 4 Sequence of motif SERIC 2 that is derived from the analysis of amino acid properties in the more conserved repeat SERIC 1.
  • Fig. 5 Testing of the glue adherence.
  • Fig. 6 Comparison of the adhesion properties of selected natural glues.
  • Fig. 7 Sequence of the synthetic gene SerA (non-coding regions incl. Ncol a Xhol cloning sites are typed in the upper case, and the cloning region in the lower case letters) and derived amino acid sequence that includes 16 repeats of the SERIC 1 motif (each second motif is underlined), flanked in the amino terminus by 23 -residue peptide and on the carboxy terminus by the RGSHHHHHH marker (RG is included in the last repeat of the pentadecapeptide motif), (sequence No. 2)
  • Fig. 8 Sequence of the synthetic gene SerB and derived amino acid sequence (bottom line). Encoded peptide includes three copies of a SERIC 2 motif followed by the nonapeptide marker RGSHHHHHH. (sequence No. 3)
  • Fig.9 Drawing of Bombyx mori silk gland showing the posterior, fibroin-secreting region (PSG), middle region producing sericins (MSG), and the anterior outlet region (ASG).
  • PSG posterior, fibroin-secreting region
  • MSG middle region producing sericins
  • ASG anterior outlet region
  • Natural biodegradable glue was identified in the silk glands of Bombyx mori as sericin 2 protein of the following amino acid sequence (shown in single-letter amino acid code).
  • Framed region corresponds to the peptide called EX9 that contains 44 repeats of the SERIC pentapeptide and is responsible for protein stickiness.
  • the region EX9 is present in the Ser2A and absent in the Ser2B sericin.
  • Sericin mix was obtained by pressing slightly the middle region (MSG) of the dissected silk gland of Bombyx mori, from which the anterior part (ASG) was removed (Fig. 9). About 3 ⁇ l of this dope were withdrawn from the gland lumen and used for the pull test of adhesion strength. The dope was smeared on the smooth base surface (0.35 cm 2 ) of a small cylinder made from the oak wood. The cylinder was immediately pressed against a wooden plate and the assembly was allowed to dry at room temperature for 24 hrs. The plate was then held in a horizontal position with the stub facing down and exposed to an increasing load (loading rate of 1 kg/min) until it detached from the plate.
  • MSG middle region
  • ASG anterior part
  • the incubation was terminated after 5 hrs by centrifugation at 600Og for 8 min.
  • the supernatant was discarded, the cells re-suspended in 6 ml lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, and 1 mg/ml lysozyme) and sonicated.
  • the lysate was centrifuged (10 OOOg/10 min) to remove cellular debris and aliquots of the supernatant containing about 50 ⁇ g total protein per 1 ml were used for electrophoresis on polyacrylamide gel (PAGE) in the presence of SDS.
  • PAGE polyacrylamide gel
  • the gel was transferred onto nitrocellulose and the recombinant protein of cca 30 kDa was detected with anti-RGS-His antibody (Qiagen). The rest of the lysate was used for affinity chromatography on the Ni-NTA resin (Qiagen). The eluted protein was dialyzed against PBS, its concentration was adjusted to 2 ⁇ g/ ⁇ l by ultrafiltration, and then it was taken to the tenacity tests. Established adhesiveness was similar to that of the starch glue (Fig. 6).
  • Synthetic gene SerB (Fig. 8), which encoded 3 repeats of the DYEKANYRSPSHR pentadecapeptide (a varient of SERIC 2 motif) and the RGS[His] 6 marker was cloned into the plasmid pET160/GW/D-TOPO (Invitrogen). (mnozstvi, viz pf. 3)£ coli strain BL21 (Invitrogen) transfected with this plasmid carrying the SerB transgene was cultured in Luria- Bertani (LB) medium at 37 °C until optical density measured at 600 nm reached value about 0.8. Transgene expression was induced with 0,8 M IPTG and terminated by centrifugation after 6 h.
  • LB Luria- Bertani
  • the sediment of bacteria was re-suspended in 3 volumes of the PBS buffer (20 mM Na 2 HPO 4 and 500 mM NaCl, pH 7.4) and incubated for 30 min at room temperature with lysozyme (Sigma) added to the concentration 0.2 mg/ml.
  • the suspension was then sonicated (150W, 6 x 10 sec) and centrifuged (5,000 g, 10 min).
  • the aliquots of supernatant were used SDS PAGE and subsequent Western analysis that revealed presence of (His) 6 in a fraction of cca 6 kDa, in accordance with the expected size of the SerB protein.
  • Raw lysate was also used to test the adhesion strength that was rather low (Fig. 6).
  • Sericin glue is useful in all areas requiring glue hardening in moist environments, especially when natural adhesives are preferred (e.g. in the wood and leather industry).
  • the sericin glues are applicable in medicine. They can be used for the fixation of denture protheses or for the attachment of plasters that cover scratches or burns. Sericin glues can also be used internally, for example for assembling broken bones and tendons.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Insects & Arthropods (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Materials For Medical Uses (AREA)
EP10747069A 2009-01-23 2010-03-02 Natural biodegradable adhesive from the silk Withdrawn EP2389387A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CZ20090029A CZ302255B6 (cs) 2009-01-23 2009-01-23 Prírodní lepivá látka
PCT/IB2010/000880 WO2010100569A2 (en) 2009-01-23 2010-03-02 Natural biodegradable adhesive from the silk

Publications (1)

Publication Number Publication Date
EP2389387A2 true EP2389387A2 (en) 2011-11-30

Family

ID=42536268

Family Applications (1)

Application Number Title Priority Date Filing Date
EP10747069A Withdrawn EP2389387A2 (en) 2009-01-23 2010-03-02 Natural biodegradable adhesive from the silk

Country Status (3)

Country Link
EP (1) EP2389387A2 (cs)
CZ (1) CZ302255B6 (cs)
WO (1) WO2010100569A2 (cs)

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CN110117613B (zh) * 2018-02-05 2022-10-11 中国科学院分子植物科学卓越创新中心 一种制备雄性不育的鳞翅目昆虫的方法及其核酸构建物
CN113563841B (zh) * 2021-07-08 2022-12-27 苏州绿豪新材料科技有限公司 一种改性植物蛋白胶黏剂及其制备方法
CN114702936B (zh) * 2022-05-18 2023-10-24 广西至善新材料科技有限公司 一种水性胶黏剂及其制备方法和应用
CN115093472B (zh) * 2022-08-25 2023-01-06 中国食品发酵工业研究院有限公司 一种具有保湿功能的丝胶蛋白肽及其制备方法和应用

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JP2720424B2 (ja) * 1994-07-04 1998-03-04 工業技術院長 セリシン含有生分解性ウレタン系発泡体及びその製造方法

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Also Published As

Publication number Publication date
WO2010100569A3 (en) 2011-01-27
WO2010100569A2 (en) 2010-09-10
CZ200929A3 (cs) 2010-08-04
CZ302255B6 (cs) 2011-01-12

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