WO2010100569A2 - Natural biodegradable adhesive from the silk - Google Patents

Abstract

The invented natural biodegradable glues include sericin 2 silk protein and therefrom derived recombinant proteins containing at least three repeats of amino acid motifs of any of the following sequences: SERIC 1 : (DX₁EKX₂KX₃NX₄X₅SPSX₆X7, )_n, where n ≥3 and X₁ = S, T X₂ = A, V X₃ = P, H X₄ = D, G X₅ = R, S X₆ = Y, H, D X₇ = K, R SERIC 2: (X₁X₂ X₃KX₄KX₅X₆X₇X₈SPX₉X₁₀X)_n, where n ≥3 and X₁ = D, P; X₂ = S, T, Y; X₃ = E, Y, D; X₄ = A, V; X₅ = P, H X₆ = N, S, A; X₇ = G, D, Y, E; X₈ = R, S X₉= S, A, K; X₁₀ = H, D, Y X₁₁ = K, R.

Description

Natural biodegradable adhesive from the silk

Technology field

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.

Background of the art

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 Britain 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. 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. Once dried, the thread (and the textiles made from it) resists moisture. The technology reveals that sericins are adhesive and differ by their solubility in water. In B. mori we found that 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

Disclosure of the invention

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. The deduced amino acid sequence of the translational product EX9, which is derived from exon 9 (Fig. 1), showed that it encodes 858 amino acid residues arranged into more than 50 copies of highly conserved SERIC motif DSEKAKPNDRSP SHK (residues not conserved in all repeats are underlined). This repetitive sequence showed remarkable similarity (35% identity over 600 amino acids) with the mussel adhesive plaque protein (Mytilus edulis) and to the repetitive sequence of adhesive protein trans-sialidase from Trypanosoma cruzi (Fig. 2). We concluded that the strength and adhesiveness of sericin 2 and blue mussel adhesive protein are mediated by the distribution of specific amino acids. Unlike the blue mussel protein, which contains a large number of tyrosine residues, the sericin 2 proteins contain few tyrosines but are rich in the polar residues.

Analysis of SERIC variability in the sticky Ser2A protein provided background for the design of a more general motif SERIC 1 that bears sticky properties (Fig. 3). Subsequent analysis of amino acid properties in the repeats led to the design of a combined "sticky" motif SERIC 2 (Fig. 4). 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 and 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 properties of sericin-type glues were compared with other natural adhesives (Fig. 5).

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. However, 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.

Description of attached figures

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).

Examples of the invention

Example 1

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.

EYEKYGEEEKYEERRTHDKFSIGKNGISAERTKSKRGERKEVEGEYEKDYERKENNGGSSEYSERERESLEKSKERYGEQSSKS FSLGKSGLKKQDNSKSYSDKEESKLEKEKKYEKKTKINNERQLDEDENERRTWGRDEQRQDDQSRDDQSRDDQSQDEETGSDD

SEQNSSNKSFNDGDASADYQTKSKKVEKNSARDKKEKEKTDTRNSDGTYKTSEREKEQSSRVNQSKGSNSRDSSESDKSGRKVN KETETYSDKDAQTSESERTQSKEKKNTAPKNKGKKGTSTETDGVTKNASKQKEfCVPKDGSKSSTNDSEGKQKNKDQSKGQKNNQ DGQDSSTNENSKKTDDNVAKKEEPNNQKREQKGKTRCGSRKTESSKAKEDRSKKSTTDKDQRDDKKDSSSKNIDKPKDGSSSDK

DSEKAKPNDRSPSHK DTEKAKPNDRSPSDK DTEKAKPNDSSPSHK DTEKAKHNDRSPSDK DTEKAKPNDRSPSHK

DTEKVKPNDRSPSHK DTEKVKPNDRSPSHK DTEKAKPNDRSPSDK DTEKAKPNDRSPSHK DTEKVKPNDRSPSYK

DTEKAKPNDRSPSDK DTEKAKPNGRSPSDK DTEKAKPNDRSPSHK DTEKVKPNDRSPSHK DTEKAKPNDRSPSDR

DTEKAKPNDRSPSDK DTEKAKPNGRSPSDK DTEKVKPNDRSPSHK DTEKAKPNDRSPSDK DTEKAKPNDRSPSDK

DTEKAKPNDRSPSHK DTEKVKPNDRSPSYK DTEKAKPNDRSPSDK DTEKAKPNGRSPSDK DTEKAKPNDRSPSHK

DTEKVKPNDRSPSHK DTEKAKPNDRSPSDR DTEKAKPNDRSPSDK DTEKAKPNGRSPSDK DTEKVKPNDRSPSHK

DTEKAKPNDRSPSDK DTEKAKPNDRSPSDK DTEKAKPNDRSPSHK DTEKVKPNDRSPSYK DTEKAKPNDRSPSDK

DTEKAKPNGRSPSDK DTEKAKPNDRSPSHK DTEKVKPNDRSPSHK DTEKAKPNDRSPSDR DTEKAKPNDRSPSDK

DTEKAKPNGRSPSDK DTEKVKPNDRSPSHK DTEKAKPNDRSPSDK DTEKAKPNDRSPSDK

KFGSNDSRARSTKAEEEHVRKSQEETHSEQREKTRSDGVTKYNDGDEHFDSDDTEKTKPNGRSPSHKDTEKAKPNDRSSSDKD TEKPFDKNIANKRPKDGSSSDKNVEQERENYKSESSRNEFENQKSAHSRYEDNGGLKEKSSQSKNYGRDEKYSEEKERSSTGK SGSNDSRARSTKAEEEHVRKSQEETHSEQRGRTRSDGATTSNDNDKQYDSDDKNNSSTKHKKTVMRSEQSDSSQNENSTSESK KFAKTDGSNKYEAESSSHKQQEARKQSNRWEKSTDGDNEESYRSESSSSSSSSSSSSRSSSSSTYTGSHDDSSEE

Example 2

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 μl 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 cm²) 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. The vertical force required to pull off the stub was taken as a measure of the tensile strength and expressed in Newtons per square centimeter (N/cm²). Value 144,75 N/cm² was established for sericin 2 collected from the silk gland.

Example 3

Gene SerA showed in Fig. 7 was designed for expression in bacteria, cloned into the Ncol and Xhol restriction sites of the pET15b expression vector (Novagen) that was subsequently used for the transformation of Escherichia coli strain BL21 (Invitrogen). The presence of insert in correct orientation was confirmed by sequencing. Bacteria were cultured at 37 °C in 50 ml portions of the Luria-Bertani (LB) broth until OD600 reached 0.8-0.9. The expression of Ser2A was then induced by addition of IPTG (isopropyl-beta-D-thiogalactopyranoside; ApplChem) to final concentration 1 mM. 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 NaH₂PO₄, 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 μg total protein per 1 ml were used for electrophoresis on polyacrylamide gel (PAGE) in the presence of SDS. 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 μg/μl by ultrafiltration, and then it was taken to the tenacity tests. Established adhesiveness was similar to that of the starch glue (Fig. 6).

Example 4

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. The sediment of bacteria was re-suspended in 3 volumes of the PBS buffer (20 mM Na₂HPO₄ 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).

Industrial applicability

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.

Claims

1. Natural biodegradable adhesive from the silk whereof structure contain at least three repeats of motif SERIC 2: (X₁X₂ X₃KX₄KX₅X₆X₇X₈SPX₉X₁₀X₁ O_n, where n >3 and

X, = D, P; X₂ = S, T, Y; X₃ = E, Y, D; X₄ = A, V;

X₅= P₅H X₆ = N, S, A; X₇= G, D, Y, E; X₈=R₅S

X₉=S₅A₅K; X₁₀=H₅D₅Y X₁₁ = K₅R

2. Natural biodegradable adhesive from the silk according to claim I₅ whereof structure contain at least three repeats of motif SERIC 1 : (DX₁EKX₂KX₃NX₄X₅SPSX₆X₇₅ )„, where n >3 and

Xi = S₅T X₂ = A₅V X_S = P₅H X₄ = D₅G

X₅ = R₅ S X₆ = Y₅ H₅ D X₇ = K₅ R

3. Natural biodegradable adhesive from the silk according to claim 2, whereof structure contain amino acid sequence of the peptide EX9.