Adhesive Polypeptides

Abstract

To provide adhesive polypeptides having excellent adhesiveness.SOLUTION: Disclosed is an adhesive polypeptide (A), which comprises polypeptide chain (y) and / or polypeptide chain (y'), where the total number of the polypeptide chains (y) and (y') is 1-100, the polypeptide chain (y) comprises 2-100 of amino acid sequence (x) which are consecutively linked where the amino acid sequence (x) is at least one selected from the group consisting of amino acid sequences containing YPG sequence and amino acid sequence containing VG sequence, where the polypeptide chain (y') is a polypeptide chain in which 0.1% to 5% amino acid residues are substituted with lysine residues on the basis of the number of the amino acid residues in the polypeptide chain (y'), and the total number of the lysine residue is 1-100.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to adhesive polypeptides. [Background technology]

[0002] Known artificial cell adhesive polypeptides have a structure in which a minimal cell adhesive amino acid sequence (X) and an auxiliary amino acid sequence (Y) are chemically bonded alternately, and the total number of amino acids contained in (Y) is 1 to 50, and the total content of glycine (Gly) and alanine (Ala) contained in (Y) is 42 to 100% by number based on the total number of amino acids in (Y) (for example, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2012-126707 Summary of the Invention [Problem to be solved by the invention]

[0004] However, even the above techniques cannot be said to be fully satisfactory in terms of adhesiveness, and there has been a demand for the development of adhesive polypeptides with better adhesiveness. An object of the present invention is to provide adhesive polypeptides with better adhesiveness. [Means for solving the problem]

[0005] The present inventors have conducted extensive research to solve the above-mentioned problems and have arrived at the present invention. Specifically, the present invention relates to an adhesive polypeptide (A) having a polypeptide chain (y) and / or a polypeptide chain (y'), wherein the total number of the polypeptide chains (y) and (y') is 1 to 100, wherein the polypeptide chain (y) is a polypeptide chain consisting of 2 to 100 consecutive amino acid residues of at least one amino acid sequence (x) selected from the group consisting of an amino acid sequence containing a YPG sequence and an amino acid sequence containing a VG sequence, and the polypeptide chain (y') is a polypeptide chain in which 0.1 to 5% of the amino acid residues in the polypeptide chain (y) are substituted with lysine residues, and the total number of the lysine residues is 1 to 100. [Effects of the Invention]

[0006] The adhesive polypeptide (A) of the present invention has the following effects. (1) Excellent adhesiveness to biological tissue. (2) Excellent adhesion to inorganic materials (metals, ceramics, etc.). (3) Excellent biocompatibility. DETAILED DESCRIPTION OF THE INVENTION

[0007] <Polypeptide chain (y)> The polypeptide chain (y) in the present invention is a polypeptide chain having 2 to 100 (preferably 3 to 30) consecutive amino acids of at least one amino acid sequence (x) selected from the group consisting of amino acid sequences containing a YPG sequence and amino acid sequences containing a VG sequence. Examples of the amino acid sequence (x) include the YPGVG sequence (1) (a sequence containing the YPG sequence and the VG sequence) shown in SEQ ID NO: 1 and the GVGYP sequence (2) (a sequence containing the VG sequence) shown in SEQ ID NO: 2. Of the above amino acid sequences (x), from the viewpoint of biocompatibility and adhesiveness, preferred are the YPGVG sequence (1) shown in SEQ ID NO: 1 and / or the GVGYP sequence (2) shown in SEQ ID NO: 2, and more preferred is the GVGYP sequence (2). In addition, among the polypeptide chains (y), preferred is (GVGYP) in which b consecutive GVGYP sequences (2) are present. b It is an array. In the above, b is preferably 3-30, and more preferably 4-15.

[0008] <Polypeptide chain (y')> The polypeptide chain (y') in the present invention is a polypeptide chain in which 0.1 to 5% (unit: % by number of amino acid residues, preferably 0.3 to 4%) of the amino acid residues in the polypeptide chain (y) have been substituted with lysine residues, and the total number of lysine residues is 1 to 100 (preferably 1 to 10, more preferably 1 to 5). Among the above polypeptide chains (y'), preferred is one in which some or all of the V (valine) residues of the polypeptide chain (y) are substituted with K (lysine), and more preferred is (GVGYP) b Some or all of the V (valine) residues in the sequence are replaced with K (lysine), and the (GVGYP)4(GKGYP)1(GVGYP)3 sequence (3) is particularly preferred.

[0009] <Adhesive Polypeptide (A)> The adhesive polypeptide (A) of the present invention has the polypeptide chain (y) and / or the polypeptide chain (y'), and the total number of the polypeptide chains (y) and (y') is 1 to 100. From the viewpoint of adhesiveness, the total number of the (y) and (y') is preferably 5 to 50.

[0010] In terms of adhesiveness and strength, the adhesive polypeptide (A) preferably has a polypeptide chain (s) in addition to the above (y) and (y'). The polypeptide chain(s) are composed of the amino acid sequence X1 AX 2 AX 3 X 4 It is a polypeptide chain in which 2 to 100 (preferably 2 to 5) sequences are linked consecutively. X 1 AX 2 AX 3 X 4 In the sequence, X 1 ~X 4 are each independently glycine, alanine or serine; A is alanine. 1 AX 2 AX 3 X 4 Among these sequences, the GAGAGS sequence (4), the GAGAGA sequence (5), and the AAAAAA sequence (6) are preferred from the viewpoint of adhesiveness and strength.

[0011] The ratio of the total number of amino acids in the (y) and (y') to the total number of amino acids in the polypeptide chain (s) among the total number of amino acids constituting the adhesive polypeptide (A) [the ratio of the total number of amino acids in the (x) and (y') in the adhesive polypeptide] 1 AX 2 AX 3 X 4 The ratio of the number of sequences between the sequence and the sum of the amino acid sequence (x) and the amino acid sequence (x') {X 1 AX 2 AX 3 X 4 From the viewpoint of adhesiveness, the ratio of the amino acid sequence (x): the total of the amino acid sequence (x) and the amino acid sequence (x')} is preferably 4:1 to 1:20, more preferably 3:1 to 1:1. Furthermore, the amino acid sequence (x') is preferably an amino acid sequence in which 20 to 60% of the amino acid residues in the amino acid sequence (x) are substituted with lysine residues, based on the total number of amino acid residues constituting (x).

[0012] Furthermore, the molecular mass of the adhesive polypeptide (A) as determined by SDS-PAGE (SDS polyacrylamide gel electrophoresis) is preferably 1 to 200 kDa, more preferably 3 to 150 kDa, and particularly preferably 5 to 100 kDa, from the viewpoints of adhesiveness and handling.

[0013] The adhesive polypeptide (A) of the present invention can be produced by known methods, such as extraction from natural products, organic synthesis (enzymatic methods, solid-phase synthesis, liquid-phase synthesis, etc.), and genetic recombination. Regarding organic synthesis, methods such as those described in "Lectures on Biochemical Experiments 1, Chemistry of Proteins IV" (published July 1, 1981, edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin Co., Ltd.) or "Continued Lectures on Biochemical Experiments 2, Chemistry of Proteins (Part 2)" (published May 20, 1987, edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin Co., Ltd.) can be applied. Regarding genetic recombination, methods such as those described in Japanese Patent No. 3338441 can be applied. Although polypeptide (A) can be obtained by extraction from natural products, organic synthesis, and genetic recombination, genetic recombination is preferred from the viewpoints of easy modification of the amino acid sequence and inexpensive mass production.

[0014] Furthermore, when the adhesive polypeptide (A) of the present invention has tyrosine (Y) as a constituent amino acid, at least one of the constituent tyrosines (Y) may be modified to dihydroxyphenylalanine in order to improve adhesiveness. The above-mentioned modification method includes, for example, the following method. (1) By adding 1000 U / mL of tyrosinase (Sigma) to a 10% by weight solution of the polypeptide and allowing it to react at 25°C for 24 hours, all of the tyrosines (Y) that make up the polypeptide can be modified to dihydroxyphenylalanine. (2) When a part of tyrosine (Y) constituting the polypeptide is to be modified to dihydroxyphenylalanine, this can be achieved, for example, by adjusting the reaction time in the above (1).

[0015] The adhesive polypeptide (A) of the present invention has excellent adhesive properties to biological tissues, excellent adhesive properties to inorganic materials (metals, ceramics, etc.), and also excellent biocompatibility. Therefore, it is applicable to various applications requiring adhesive properties, such as the liniment (α) described below, particularly liniment for wound healing and liniment for medical devices. It is also suitable as an adhesive when applied.

[0016] <Coating Agent (α)> The coating agent (α) of the present invention comprises the adhesive polypeptide (A). The adhesive polypeptide (A) may be used as is in the coating agent (α). It may also be diluted with an aqueous medium (for example, water), in which case the concentration of the adhesive polypeptide (A) is preferably 1 to 20% by weight, more preferably 2 to 15% by weight, based on the weight of the application agent.

[0017] <Medical Devices> The medical device of the present invention is formed by coating the coating agent (α) on an inorganic material, or by bonding inorganic materials together with the coating agent (α), for example. Specific examples include catheters, stents, pacemakers, sensors, dialysis machines, artificial joints, artificial bones, etc. Also, the present invention is preferably used as a surface coating agent for these implant materials. It is suitable. [Example]

[0018] The present invention will be further described below with reference to examples and comparative examples, but the present invention is not limited thereto. Unless otherwise specified, % means % by weight and parts means parts by weight.

[0019] <Production Example 1> Production of SELP8KY1 Plasmid pPT0345 encoding SELP8K was prepared according to the method described in the Examples of Japanese Patent No. 4088341. pPT0345 was cleaved with BamHI and EcoRV to prepare an artificially synthesized (synthesized by Thermo) plasmid pRS001 encoding SELP8KY1 having BamHI and EcoRV cleavage sites at both ends. The constructed plasmid pRS001 was transformed into Escherichia coli to obtain a SELP8KY1-producing strain. An overnight culture of the SELP8KY1-producing strain grown at 30°C was used to inoculate 50 ml of LB medium in a 250 ml flask. Kanamycin was added to a final concentration of 50 μg / ml, and the culture was cultured at 30°C with stirring (200 rpm). When the culture reached an OD600 of 0.8 (using a Shimadzu UV1700 spectrophotometer), 40 ml was transferred to a flask preheated to 42°C and cultured at the same temperature for approximately 2 hours. The culture was cooled on ice, and the OD600 of the culture was measured. E. coli was collected by centrifugation. To isolate the polypeptide from the collected E. coli, the cells were lysed by ultrasonic disruption (4°C, 30 seconds x 10 times). The protein produced by this E. coli was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride membrane. Western blot analysis was then performed using rabbit anti-SELP8KY1 antibody as the primary antibody and anti-rabbit IgG HRP-labeled antibody (GE Healthcare) as the secondary antibody. The apparent molecular mass of the product was approximately 80 kDa. Therefore, it was found that the SELP8KY1-producing strain produced SELP8KY1 with an apparent molecular mass of 80 kDa and reactivity with rabbit anti-SELP8KY1 antibody.

[0020] Purification of SELP8KY1 The SELP8KY1 obtained above was purified from the E. coli biomass by cell lysis, centrifugation to remove insoluble debris, and affinity chromatography to obtain a polypeptide (A-1) (SELP8KY1) with a molecular mass of approximately 80 kDa.

[0021] Identification of SELP8KY1 The resulting polypeptide (A-1) was identified by the following procedure. Western blot analysis was performed using rabbit anti-SELP8KY1 antibody and rabbit anti-6xHis antibody (Roland) against the 6xHis tag in the C-terminal sequence. A protein band with an apparent molecular mass of 80 kDa showed antibody reactivity with each antibody. Amino acid analysis of the resulting polypeptide revealed that the product was enriched in glycine (43.7%), alanine (12.3%), serine (5.3%), proline (11.7%), valine (11.2%), and tyrosine (11.1%). The product also contained 1.5% lysine. Table 1 below shows the correlation between the composition of the purified product and the predicted theoretical composition inferred from the synthetic gene sequence. Therefore, it was confirmed that polypeptide (A-1) is a polypeptide having 13 (GVGYP)4GKGYP(GVGYP)3 sequences (3) and 12 (GAGAGS)4 sequences (7), which are chemically bonded alternately, and a (GAGAGS)2 sequence (8) chemically bonded to sequence (9).

[0022] [Table 1]

[0023] <Production Example 2> Polypeptide (A-2) having a molecular mass of approximately 80 kDa and sequence (10) was obtained in the same manner as in Production Example 1, except that "plasmid pRS002 encoding SELP8KY2" was used instead of "plasmid pRS001 encoding SELP8KY1." Using the same identification method as described in Production Example 1, it was confirmed that polypeptide (A-2) was a polypeptide having sequence (10) in which 13 (GVGYP)4GKGYP(GVGYP)3 sequences (3) and 12 (GAGAGA)4 sequences (11), which were chemically bonded alternately, and to which a (GAGAGA)2 sequence (12) was chemically bonded.

[0024] <Production Example 3> Polypeptide (A-3) having a molecular mass of approximately 80 kDa and sequence (13) was obtained in the same manner as in Production Example 1, except that "plasmid pRS003 encoding SELP8KY3" was used instead of "plasmid pRS001 encoding SELP8KY1." Using the same identification method as described in Production Example 1, it was confirmed that polypeptide (A-3) was a polypeptide having sequence (13) in which 13 (GVGYP)4GKGYP(GVGYP)3 sequences (3) and 12 (AAAAAA)4 sequences (14) were chemically bonded alternately to a (AAAAAA)2 sequence (15) chemically bonded thereto.

[0025] <Production Example 4> Polypeptide (A-4) having a molecular mass of approximately 80 kDa and sequence (16) was obtained in the same manner as in Production Example 1, except that "plasmid pPT0345 encoding SELP8K" was used instead of "plasmid pRS001 encoding SELP8KY1." Using the same identification method as described in Production Example 1, polypeptide (A-4) was confirmed to be a polypeptide having sequence (16), which has 13 (GVGVP)4GKGVP(GVGVP)3 sequences (3) and 12 (GAGAGS)4 sequences (7), which are chemically bonded alternately, and to which a (GAGAGS)2 sequence (8) is chemically bonded.

[0026] <Production Example 5> The prepared polypeptide (A-1) was dissolved in 50 mM potassium phosphate buffer (pH 6.5) to a concentration of 10% by weight. Tyrosinase (Sigma) was added to this solution to a final concentration of 1000 U / mL, and the mixture was allowed to react at 25°C for 0.5, 2, 12, or 24 hours to convert the tyrosine in the polypeptide (A-1) chain to dihydroxyphenylalanine. The reacted solution was purified by affinity chromatography to obtain polypeptides (A-5) to (A-8) (17).

[0027] <Production Example 6> The prepared polypeptide (A-2) was dissolved in 50 mM potassium phosphate buffer (pH 6.5) to a concentration of 10% by weight. Tyrosinase (Sigma) was added to this solution to a final concentration of 1000 U / mL, and the reaction was allowed to proceed at 25°C for 0.5, 2, 12, or 24 hours to convert the tyrosine in the polypeptide (A-2) chain to dihydroxyphenylalanine. The reaction solution was purified by affinity chromatography to obtain polypeptides (A-9) to (A-12) (18).

[0028] <Production Example 7> The prepared polypeptide (A-3) was dissolved in 50 mM potassium phosphate buffer (pH 6.5) to a concentration of 10% by weight. Tyrosinase (Sigma) was added to this solution to a final concentration of 1000 U / mL, and the reaction was allowed to proceed at 25°C for 0.5, 2, 12, or 24 hours to convert the tyrosine in the polypeptide (A-3) chain to dihydroxyphenylalanine. The reaction solution was purified by affinity chromatography to obtain polypeptides (A-13) to (A-16) (19).

[0029] Example 1: Evaluation of in vitro adhesive strength Polypeptide (A-1) was dissolved in pure water to a concentration of 10% by weight to prepare a liniment. Pig skin was fixed to the measurement site of a hydraulic universal testing machine (UTM), and 100 μL of the liniment was applied to the skin. After leaving the skin at 37°C for 10 minutes, the adhesive strength was evaluated. The results are shown in Table 2.

[0030] <Examples 2 to 15> The adhesive strengths of Examples 2 to 15 were evaluated in the same manner as in Example 1, except that polypeptides (A-2), (A-3), and (A-5) to (A-16) were used instead of polypeptide (A-1). The results are shown in Table 2.

[0031] <Comparative Example 1> The adhesive strength of Comparative Example 1 was evaluated in the same manner as in Example 1, except that polypeptide (A-4) was used instead of polypeptide (A-1). The results are shown in Table 2.

[0032] <Comparative Example 2> The adhesive strength of Comparative Example 2 was evaluated in the same manner as in Example 1, except that fibrin glue (Beliplast P Combicet, manufactured by CSL Behring) was used instead of polypeptide (A-1). The results are shown in Table 2.

[0033] [Table 2]

[0034] Example 16: Evaluation of in vivo hemostatic activity and inflammatory response The back of a white rabbit was depilated using clippers and a depilatory agent, and a 5 cm wound was made with a scalpel to prepare a test specimen. 500 mg of polypeptide (A-1) was applied to the wound of the test specimen and left to stand for 10 minutes. After 10 minutes, gauze was placed on the wound and gently pressed against it. The difference in the weight of the gauze before and after the test was used to evaluate the adhesiveness and hemostatic performance. The test specimens were also observed for the occurrence of inflammation as an evaluation of biocompatibility. Observations included the presence and extent (spread, thickness, etc.) of bleeding and discoloration in the tissue surrounding the test specimen, which were observed with the naked eye. The results are shown in Table 3. In the table, inflammatory reactions are indicated by + if present and - if absent.

[0035] <Examples 17 to 30> The hemostatic performance of Examples 17 to 30 was evaluated in the same manner as in Example 16, except that polypeptides (A-2), (A-3), and (A-5) to (A-16) were used instead of polypeptide (A-1). The results are shown in Table 3.

[0036] <Comparative Example 3> The hemostatic performance of Comparative Example 3 was evaluated in the same manner as in Example 8, except that polypeptide (A-4) was used instead of polypeptide (A-1). The results are shown in Table 3.

[0037] <Comparative Example 4> Except for using fibrin glue instead of polypeptide (A-1), the same procedure as in Example 16 was carried out to evaluate the hemostatic performance of Comparative Example 4. The results are shown in Table 3.

[0038] <Comparative Example 5> The hemostatic performance of Comparative Example 5 was evaluated in the same manner as in Example 16, except that the wound was sutured using suture thread instead of polypeptide (A-1). The results are shown in Table 3.

[0039] [Table 3]

[0040] <Example 31> Adhesion test to metal Polypeptide (A-1) was dissolved in pure water to a concentration of 10 wt % to prepare a coating agent. 5 μL of the prepared adhesive was dropped onto the surfaces of metal plates (aluminum, iron, and titanium plates) and ceramic plates, and then dried under reduced pressure at room temperature for 30 minutes. Next, each plate was washed in distilled water for 30 minutes at 45 rpm, and then dried again at room temperature for 30 minutes under reduced pressure. After drying, each plate was immersed in 0.25% Coomassie Brilliant Blue (CBB) R-250 solution (Bio-Rad) for 1 hour and then washed several times with distilled water. The blue areas stained with CBB were photographed, and the stained area was measured using ImageJ image analysis. If the polypeptide (A-1) adhered to the surface of each plate, a blue area was confirmed by CBB staining. The results are shown in Table 4. The "stained area (relative value)" in Table 4 is expressed as a relative ratio when the stained area of ​​Comparative Example 7 was set to 1.00.

[0041] <Examples 32 to 45> The adhesion to metal of Examples 32 to 45 was evaluated in the same manner as in Example 31, except that polypeptides (A-2), (A-3), and (A-5) to (A-16) were used instead of polypeptide (A-1). The results are shown in Table 4.

[0042] <Comparative Example 6> The adhesion to metal of Comparative Example 6 was evaluated in the same manner as in Example 31, except that polypeptide (A-4) was used instead of polypeptide (A-1). The results are shown in Table 4.

[0043] <Comparative Example 7> Except for using fibrin glue instead of polypeptide (A-1), the same procedure as in Example 31 was carried out to evaluate adhesion to metal in Comparative Example 7. The results are shown in Table 4.

[0044] <Comparative Example 8> Except for using bovine serum albumin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) instead of polypeptide (A-1), the same procedure as in Example 31 was performed to evaluate adhesion to metal in Comparative Example 8. The results are shown in Table 4.

[0045] [Table 4]

[0046] The results in Tables 1 to 4 show that the adhesive polypeptide (A) of the present invention has superior adhesiveness to biological tissues and also superior adhesiveness to inorganic materials (metals, ceramics, etc.) compared to the comparative polypeptides. It also has excellent biocompatibility. [Industrial Applicability]

[0047] It has been found that the adhesive polypeptide (A) of the present invention has high adhesive properties to biological tissues, metals, ceramics, etc., and further has high hemostatic properties. Therefore, the polypeptide and coating agents and adhesives containing the same can be used as adhesives and hemostatic agents for biological tissues. In addition, by using the polypeptide as a surface coating agent for implant materials such as sensors and medical devices (artificial bones, stents, etc.), the biocompatibility of these devices can be improved.

Claims

1. An adhesive polypeptide having a polypeptide chain (y) and / or a polypeptide chain (y'), wherein the total number of the polypeptide chains (y) and the polypeptide chains (y') is 1 to 100, the polypeptide chain (y) is a polypeptide chain comprising 2 to 100 consecutive amino acids of at least one amino acid sequence (x) selected from the group consisting of an amino acid sequence containing a YPG sequence and an amino acid sequence containing a VG sequence, The amino acid sequence (x) is a YPGVG sequence (1) which is the amino acid sequence shown in SEQ ID NO: 1 and / or a GVGYP sequence (2) which is the amino acid sequence shown in SEQ ID NO: 2, the polypeptide chain (y') is a polypeptide chain in which 0.1 to 5% of the amino acid residues in the polypeptide chain (y) are substituted with lysine residues, based on the number of amino acid residues in the polypeptide chain (y), and the total number of the lysine residues is 1 to 100; The adhesive polypeptide (A) further comprises a polypeptide chain (s) in which 2 to 100 consecutive amino acid sequences of the amino acid sequence X1AX2AX3X4 are linked together, and the X1AX2AX3X4 sequence is (GAGAGS), (GAGAGA) or (AAAAAA).

2. In the adhesive polypeptide, 1 AX 2 AX 3 X 4 The ratio of the number of sequences to the total of the amino acid sequence (x) and the amino acid sequence (x') {X 1 AX 2 AX 3 X 4 2. The adhesive polypeptide according to claim 1, wherein the amino acid sequence (x): the total of the amino acid sequence (x) and the amino acid sequence (x') is 4:1 to 1:20, and the amino acid sequence (x') is an amino acid sequence in which 20 to 60% of the amino acid residues in the amino acid sequence (x) are substituted with lysine residues, based on the number of amino acid residues in the amino acid sequence (x).

3. 3. The adhesive polypeptide according to claim 1, wherein the molecular mass of the adhesive polypeptide determined by SDS-PAGE (SDS polyacrylamide gel electrophoresis) is 1 to 200 kDa.

4. 4. The adhesive polypeptide according to claim 1, wherein at least one of the tyrosines (Y) constituting the adhesive polypeptide is modified to dihydroxyphenylalanine.

5. A coating agent (α) comprising the adhesive polypeptide (A) according to any one of claims 1 to 4.

6. 6. The coating agent according to claim 5, wherein the concentration of the adhesive polypeptide (A) is 1 to 20% by weight based on the weight of the coating agent.

7. A medical device coated with the coating agent (α) according to claim 5 or 6.

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