CZ200929A3 - Natural biologically degradable protein adhesive - Google Patents

Abstract

A natural biodegradable protein glue that contains a protein whose structure contains at least three repeats of 15 amino acids. This sequence represents the adhesive motif of a protein called sericin 2, which is a product of the Ser2 silkworm gene (Bombyx mori).

Description

PV 2004 · 13 Natural biodegradable protein adhesive

Technical field

Identification of the amino acid sequence of sericin 2A that acts on its adhesive. Utilization of natural sericin and recombinant proteins with the same or similar amino acid sequence as adhesives.

BACKGROUND OF THE INVENTION

Natural adhesives have been used in various human cultures since antiquity and their functionality can be verified even after millennia. The best-known glue of animal origin is glue (known from furniture in Egyptian graves and has been proven to have been used by ancient Sumerians), which results from long-term cooking of bones, hides or hooves of horses or cattle. Another commonly used glue is casein, based on the precipitation of dairy products in alkaline water and resistant to moisture and aging better than glue. Of the vegetable adhesives, starch, cellulose and gum are best known. Starch is currently one of the most widely used adhesives ever and is most commonly made from corn, tapioca, sago, wheat or potatoes. Natural adhesives are also produced from some bacteria. A disadvantage of commonly used natural adhesives is the relatively low strength and low moisture resistance. An exception among natural adhesives is clam adhesive, which excels in strength and can solidify even under water.

The first known natural glue was glue made from fish. Since then, many patents have been issued for natural rubber, bone, fish, starch and milk casein adhesives. For example, since 1896, it has been known to prepare degenerate starch, which is said to be almost equal in strength to glue.

Butterfly cocoons are a potential source of natural glue. Natural silk in cocoon butterflies, such as silkworms, consists of two axial filaments, which are glued together in a single fiber by proteins called sericins, which are located in the package and are up to 9 types. In the production of raw silk, the cocoons are soaked in hot water, thereby dissolving the top layers of sericins and releasing the sticky fiber from the cocoon wall. Several fibers are held together in a spinning machine, due to the sericins, adhere closely to each other, and are stuck together in a raw silk thread, which is wetly pulled through the loop to the desired diameter. It is clear from this technology that some sericins are sticky.

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SUMMARY OF THE INVENTION

Sericin, which is responsible for tackiness and is useful for the preparation of natural biodegradable adhesive, has been identified and characterized.

The sticky sericin fraction can be obtained from the caterpillar glands of caterpillars 3.-5. the day of the last larval instar. At that time, two proteins accumulate in the anterior third of the middle section of the glands, whose extract has strong tack properties.

These proteins were isolated; The sequence of the silkworm Ser2 gene was determined using biochemical methods and molecular biology techniques and was shown to encode these proteins. The production of two proteins, called Sericin 2A and 2B, is due to different splicing of the primary transcript. If all the exons are retained in the mRNA, sericin 2A is produced which contains 1740 amino acids and has a molecular size of about 220 kDa. Sericin 2B contains only 882 amino acids and its size is about 130 kDa because the respective mRNA lacks the 9th exon. Analysis of theoretically derived translation products of the Ser2 gene showed that the 9th exon encodes a 858 amino acid protein region named EX9. Usek contains 44 repeats of the 15 amino acid motif. Based on the analysis of the motif variability in the region encoded by exon 9 and the analysis of the properties of the individual amino acids, a general sequence motif responsible for the tackiness of sericins 2 and called SERIC 1 was derived.

Similarity was observed to the motif responsible for the tackiness of the protein products of the mussel clam (Mytilus edulis) and the trypanosome adhesive protein. Subsequently, a more general repeat sequence sequence was derived corresponding to these sequences and called SERIC 2 Unlike a clam protein that contains a large amount of tyrosine residues, sericin 2 is relatively low in tyrosine. Conversely, it is rich in polar amino acid residues.

Sticky are those peptide and protein regions that contain at least three repeated 15 amino acid sequences with different amino and carboxyl-terminal extensions.

The protein-sericin 2A sticky region that is encoded by exon 9, called EX 9:

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The general repeating amino acid sequence forming the sticky region of protein-sericin 2A, which is encoded by exon 9, called SERIC 1:

DX EKX _{1 2 3} KX ₄ NX X5SPSX ₆ X ₇ wherein

X1 = S, T

X ₂ = A, V

X ₃ = P, H

X 4 = D, G x ₅ = R, p

Χ ,, - Y. H, D

X ₇ = K, R

A general amino acid sequence that contains proteins forming biodegradable adhesives called SERIC 2:

(XiX ₂ X ₃ KX4KX ₅ X ₆ X7X ₈ SPX ₉ XioX ₁ i) _n where

X = D, P; X ₂ = S, T, Y; X 3 = E, Y, D; X ₄ = A, V; X 5 = P, H X ₆ = N, S, A; X ₇ = G, D, Y, E; X g = R, S X ₉ = S, A, K; X 10 = H, D, Y X n = K, R

Sericin glue can be prepared in three ways: by processing silkworm glands; gently washing sericins from cocoon; expressing a portion of the natural gene and / or the synthetic gene in a suitable vector, e.g., in Pichia pastoris. The first option was used to prepare the material for adhesion strength tests of glued surfaces. The latter was not possible, but the tackiness of the raw silk fibers in cocoon processing suggests that a suitable technology could be developed. Exon 9 of the Ser2 gene or a synthetic gene encoding a sequence of type SERIC 1 or SERIC 2 may be used to prepare the recombinant sericin adhesive.

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Biodegradable protein adhesives based on sericin have many uses. Its advantage is solidification in a humid environment. Therefore, they can be used wherever other natural water-soluble adhesives have been used (eg wood industry, leather production). Water-soluble adhesives are more environmentally friendly and healthier than synthetic solvents soluble in organic solvents, but their setting is often slow because it depends on the evaporation of water. Sericin adhesives have a great advantage over starch adhesives and glues in that they cure quickly even in humid environments and are therefore used in wet plants. Because it does not induce immune reactions or allergies (until the discovery of synthetic fibers, silk has been used as surgical threads), the sericin adhesive is suitable for application to the body surface, e.g. Recombinant sericin adhesives of known composition can also be used for bonding implants to damaged tissues after biocompatibility has been verified. Since a thin layer of adhesive forms a film close to the substrate, it is envisaged to use sericin adhesives as a cover for abrasions and larger wounds and burns. Recombinant sericin adhesives of precisely known compositions will be particularly important for use in medicine.

The properties of the sericin adhesive were compared with conventional natural adhesives by testing. Sericin glue had a significantly higher efficiency than starch glue, but lower than bone glue. However, it should be taken into account that raw preparations of sericin glue were used for the tests, but commercial glues have been optimized for centuries. A great advantage of sericin adhesive is its setting in a humid environment. Both glue and starch adhesives only solidify when the water in which the sticky components are dissolved is evaporated. The solidification rate of the sericin adhesive is remarkable - the investigated sericin fraction solidifies within a minute after pressing the glued surfaces together.

BRIEF DESCRIPTION OF THE DRAWINGS

Giant. 1: amino acid sequence encoded by exon 9 of the Ser2 gene. SERIC l.Obr. 2: amino acid sequence SERIC 2. FIG. 3: amino acid sequence EX 9. FIG. 4: amino acid sequence of secreted Ser2A protein (after cleavage of signal peptide). The repetitive region encoded by exon 9 is framed. It is key to the adhesive properties of the protein. Giant. 5: Schematic diagram of adhesive efficiency testing FIG. 6: comparison of the adhesive properties of natural adhesives. Giant. 7: Prepared silkworm gland ,; AMSG is a region with maximum production of Ser2 protein.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Natural biodegradable protein glue derived from silkworm glands such as sericin 2A with the amino acid sequence;

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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

dtdktfdknidnkrpkdgsssdknveqerenyksessrnefenqksahsryedngglkekssqsknygrdekyseekerss tgkfgsndsrarstkaeeehvrksqeethseqrektrsdgvtkyndgdehfdsddtektkpngrspshkdtekakpndrsssd kdtekpfdkntankrpkdgsssdknveqerenyksessrnefenqksahsryedngglkekssqsknygrdekyseekersst gksgsndsrarstkaeeehvrksqeethseqrgrtrsdgattsndndkqydsddknnsstkhkktvmrseqsdssqnenstse skkfaktdgsnkyeaessshkqqearkqsnrvvekstdgdneesyrsessssssssssssrssssstytgshddssee

The 15 amino acid repeating region of the protein corresponding to the general sequence responsible for the adhesive properties is framed.

Example 2

The prepared glander can be directly used to obtain the sericin proteins for tack testing. After cutting the thin lead (ASG), the contents of its central part of the MSG are pushed out with a slight pressure, and 2-3 μΐ are applied to the smooth surface of the base (0.35 cm ² ) of the beech roller. The roller is immediately pressed against the wooden underlay with this surface. After 24 hours, a container is attached to the roller adhered to the base plate (Fig. 5), into which water is added at a rate of 1 ml / sec until the roller is peeled off. The resulting weight of added water required to tear off the roller is converted to cm ^{2 of} bonded area. The force required to tear off the roller was calculated from the water volume and the weight of the vessel and was 144.7546 N / cm ² .

Industrial applicability

Natural biodegradable protein adhesives are useful in medicine; surgery, dentistry, dermatology. In industry sectors where other natural water-soluble adhesives are still in use (eg wood industry, leather production)

Claims (3)

A natural biodegradable protein glue whose structure comprises an amino acid sequence designated SERIC 2. The natural biodegradable proteinaceous adhesive of claim 1 wherein the structure comprises a repeating sequence of amino acids designated SERIC 1. The natural biodegradable proteinaceous adhesive of claim 2, wherein the structure comprises an amino acid sequence designated EX 9.