An anionic peptide with adhesion-enhancing properties

Abstract

This invention discloses an anionic peptide with adhesion-enhancing properties. The tandem repeating sequence of alternating aspartic and glutamic acid in the anionic peptide can copolymerize with cationic adhesive peptides through covalent bonds and electrostatic interactions, thereby enhancing cohesion and shortening curing time. This anionic peptide with adhesion-enhancing properties can improve the microscopic and macroscopic adhesion and adsorption capacity of cationic adhesive peptides, and can be applied in fields such as biomanufacturing and biomedicine.

Description

Technical Field

[0001] This invention belongs to the field of biomimetic adhesion technology, specifically relating to an anionic peptide with adhesion-enhancing properties. Background Technology

[0002] The waterproof protein (PC) secreted by the sand worm is a polyelectrolyte with opposite charges. The polyanionic peptides and polycationic peptides can increase the cohesive force of PC and shorten the curing time through electrostatic interactions and covalent bonds. At the same time, L-3,4-dihydroxyphenylalanine (Dopa) in PC can enhance the adhesion.

[0003] Literature review revealed that the microscopic adhesion of both expressed and synthesized adhesive materials was not particularly strong. Atomic force microscopy (AFM) experiments showed that a colloidal probe with a 5 μm radius glass bead tip exhibited a maximum adhesion of 201 ± 36 nN / μm to the tandem polypeptide of mussel adhesion protein 5 (Mfp5) oligomers. The microscopic adhesion of short model peptides inspired by mussel adhesion protein 3 (Mfp3) and Mfp3 variant proteins ranged from 5.17 to 12.85 mN / m and 37.68 to 95.53 mN / m, respectively. Binding the analyte to a colloidal probe using aminopropyltriethoxysilane and glutaraldehyde revealed that the adhesion of *E. coli*-expressed *Cypripedium acicularis* cp19k to a mica substrate reached 0.88 ± 0.24 mJ / m. 2 Cell-Tak achieves an adhesion strength of 1.54 ± 0.59 mJ / m on its surface. 2 Therefore, redesigning peptide complexes with stronger adhesion by analyzing the sequence characteristics of Mfp is a challenging problem that needs to be solved by those skilled in the art. Summary of the Invention

[0004] The purpose of this invention is to provide an anionic peptide with adhesion-enhancing properties.

[0005] An anionic peptide with adhesion-enhancing properties has the amino acid sequence Dopa-DEDE-Dopa-DEDE-Dopa-DEDE-Dopa-DEDE-Dopa-DEDE-Dopa, where Dopa is L-3,4-dihydroxyphenylalanine. The anionic peptide was prepared by Nanjing Peptide Valley Biotechnology Co., Ltd., CAS: TG-LG-11912.

[0006] The tandem repeating sequence of alternating aspartic acid and glutamic acid in the anionic peptide can copolymerize with the cationic adhesive peptide through covalent bonds and electrostatic interactions to enhance cohesion and shorten curing time.

[0007] The cationic adhesion peptide was prepared by Nanjing Peptide Valley Biotechnology Co., Ltd., CAS: TG-LG-11810; its amino acid sequence is: KYGGKWGGK-Dopa-K-Dopa-K-Dopa-GGKWGGK-Dopa-K-Dopa-K-Dopa-GGKWGGKY; the combination of the anionic peptide with adhesion-enhancing effect and the cationic peptide with adhesion properties can improve the adhesion strength and adsorption capacity.

[0008] Preferably, the adhesion force is maximized when the ratio of anionic peptides with adhesion-enhancing effects to cationic adhesive peptides is 1:1.

[0009] Application of the anionic peptide with adhesion-enhancing properties in the preparation of medical adhesives.

[0010] Application of the anionic peptide with adhesion-enhancing properties in the preparation of biochips.

[0011] The beneficial effects of this invention are as follows: The anionic peptide with adhesion-enhancing properties of this invention can enhance the microscopic and macroscopic adhesion and adsorption capacity of cationic adhesive peptides. The biomimetic adhesive of this invention with the above-mentioned properties can be applied in fields such as biomanufacturing and biomedicine. Attached Figure Description

[0012] Figure 1 Adhesion pressure analysis was performed on cationic adhesive peptides with different curing times, anionic peptides with adhesion-enhancing effects, and mixtures of the two in different proportions.

[0013] Figure 2 The atomic force spectrum consists of cationic adhesion peptides, anionic peptides with adhesion-enhancing effects, and a 1:1 mixture of the two.

[0014] Figure 3 This study analyzed the adsorption capacity of cationic adhesive peptides and anionic peptides with adhesion-enhancing effects. Detailed Implementation

[0015] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0016] Example 1: Analysis of the enhancing effect of anionic peptides with adhesion-enhancing properties on the shear pressure of cationic adhesive peptides

[0017] 1. Samples of anionic peptides (Nanjing Peptide Valley Biotechnology Co., Ltd., CAS: TG-LG-11912) and cationic adhesion peptides (Nanjing Peptide Valley Biotechnology Co., Ltd., CAS: TG-LG-11810) with adhesion-enhancing effects were respectively prepared into polypeptide solutions with a mass concentration of 5% using ultrapure water.

[0018] 2. For the analysis of adhesion pressure of a single sample, 1.4 μl of peptide solution was dropped onto the surface of a 1 cm × 1.5 cm glass slide. Another glass slide of the same size was quickly placed on top and fixed with clips. The slides were cured for 0.5 h, 6 h, and 24 h, respectively. For the analysis of the interaction between anionic and cationic adhesive peptides with adhesion-enhancing effects, 1.4 μl of peptide solution was dropped onto the surface of a 1 cm × 1.5 cm glass slide, ensuring that the ratio of anionic to cationic adhesive peptides was 1:2, 1:1, and 2:1, respectively. The two glass slides were then quickly placed together and cured for 0.5 h, 6 h, and 24 h, respectively.

[0019] 3. The shear pressure of the cured peptide was measured using a tensile strength testing machine. The results showed that the anionic peptide significantly enhanced the adhesion of the cationic adhesive peptide, and the adhesion enhancement effect was most significant when the ratio of the two was 1:1. Figure 1 ).

[0020] Example 2: Analysis of the adsorption capacity of anionic and cationic peptides

[0021] Sample preparation:

[0022] 1. Prepare a pH 6.0 PBS buffer solution and autoclave it.

[0023] 2. Prepare a polypeptide solution of 1 mg / ml using PBS for both samples and store at 4°C.

[0024] Measurement steps:

[0025] 1. Place the injection tube in ultrapure water, turn on the peristaltic pump, set the flow rate to 50 μl / min, start the injection and observe the baseline until it stabilizes.

[0026] 2. Acquisition-restart measurement: After stabilizing in air for 5 minutes and achieving good frequency overlap, start passing through pH 6.0 PBS buffer and start timing for 10-15 minutes from the time the liquid response signal is observed.

[0027] 3. Acquisition-restart measurement: If there is no significant difference in the octave, start the test. The baseline equilibration time should not be less than 10 minutes.

[0028] 4. Cationic peptides adhere to and adsorb onto the chip until baseline equilibrium is reached.

[0029] 5. Rinse with pH 6.0 PBS buffer for 10 min.

[0030] 6. Anionic peptides adsorb onto cationic adhesive peptides until baseline equilibrium is reached.

[0031] 7. Acquisition-stop: Store data, stop the pump, and adjust the flow rate to 300 μl / min to flush the system.

[0032] The results showed that the anionic peptides with adhesion-enhancing properties had a significant adsorption capacity for cationic adhesives, reaching up to 155 ng / cm³. 2 ( Figure 2 ).

[0033] Example 3: Analysis of the microscopic adhesion of cationic and anionic peptides

[0034] The microscopic adhesion forces of cationic adhesive peptides, anionic peptides with adhesion-enhancing effects, and a 1:1 mixture of the two were measured using atomic force microscopy. The peptides were prepared as a 0.5% aqueous solution, and the probe pressure on the sample was set to 2 nN. Surface adhesion force measurements were conducted in atmospheric conditions at 25°C and 28% relative humidity. The instrument used was a Bruker FASTSCANBIO atomic force microscope (Germany), with a FASTSCAN-C probe. The probe tip was a triangular Si3N4 with a radius of curvature of 5 nm, an elastic modulus of 0.8 N m⁻¹, and a resonant frequency of 300 kHz.

[0035] The results showed that anionic peptides (enhancers) with adhesion-enhancing effects could increase the microscopic adhesion of cationic adhesive peptides by approximately 60%, from the original 9.28 nN to 14.77 nN. Figure 3 ).

[0036] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An anionic peptide with adhesion-enhancing properties, characterized in that, Its amino acid sequence is Dopa-DEDE-Dopa-DEDE-Dopa-DEDE-Dopa-DEDE-Dopa-DEDE-Dopa-DEDE-Dopa, where Dopa is L-3,4-dihydroxyphenylalanine; The tandem repeating sequence of alternating aspartic acid and glutamic acid in the anionic peptide can copolymerize with the cationic adhesive peptide through covalent bonds and electrostatic interactions to enhance cohesion and shorten curing time. The combination of anionic peptides with adhesion-enhancing properties and cationic peptides with adhesion properties can improve adhesion strength and adsorption capacity.

2. The application of the anionic peptide with adhesion-enhancing properties as described in claim 1 in the preparation of medical adhesives.

3. The application of the anionic peptide with adhesion-enhancing effect as described in claim 1 in the preparation of biochips.

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