A streptavidin containing a long-chain mercapto arm and a preparation method thereof

Abstract

The present invention discloses a streptavidin containing a long-chain mercapto arm and a preparation method thereof, belonging to the field of biomedicine. The preparation method of the streptavidin containing a long-chain mercapto arm comprises the following steps: 1) dissolving the cysteine-type recombinant core streptavidin in an appropriate buffer solution, exposing cysteine residues under the action of an excessive reducing agent, and then separating and removing the excessive reducing agent; 2) reacting the solution obtained in step 1) with an excessive cross-linking agent, and then separating and removing the excessive cross-linking agent; 3) exposing sulfhydryl groups in the solution obtained in step 2) under the action of an excessive reducing agent, and separating and removing the excessive reducing agent, thereby obtaining the streptavidin containing a long-chain mercapto arm. The streptavidin containing a long-chain mercapto arm prepared in this application solves the problem of low bonding efficiency between the cysteine-type recombinant core streptavidin and the carrier.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a streptavidin containing a long-chain mercapto arm and a preparation method thereof. Background Art

[0002] Streptavidin (SA) is a tetrameric protein derived from Streptomyces avidinii, and has similar biological properties to avidin. It can specifically bind to four molecules of biotin. Since SA does not contain glycosyl groups, the non-specific binding level of SA is much lower than that of avidin. Based on the high affinity between SA and biotin and the multi-stage amplification effect, the SA-biotin system has been widely used in biotechnological fields such as enzyme-linked immunosorbent assay, immunohistochemistry, time-resolved immunofluorescence technique, quantitative PCR, single-stranded DNA preparation, biomolecule purification, monoclonal antibody preparation, etc.

[0003] Compared with natural streptavidin, recombinant core streptavidin (r-cSA) removes the amino acid sequences irrelevant to activity and only retains the core 127aa sequence. Its stability, solubility, etc. are superior to natural SA. Cysteine-type recombinant core streptavidin adds one cysteine at the C-terminus of r-cSA, and its mercapto group can be covalently bonded to the carrier directionally. It has now become one of the most commonly used streptavidins in this field. However, affected by the steric hindrance of the mercapto group, the bonding efficiency of cysteine-type recombinant core streptavidin is relatively low. Therefore, it is necessary to develop a mercapto-modified recombinant streptavidin with small steric hindrance. Summary of the Invention

[0004] One object of the present invention is to provide a streptavidin containing a long-chain mercapto arm, and its structure is shown in Formula I.

[0005]

[0006] Wherein,

[0007] SA is cysteine-type recombinant core streptavidin;

[0008] E is selected from: C1-C 10 linear alkyl, C3-C 10 branched alkyl, cyclic alkyl with 3-8 carbon atoms, aryl with 6- 10 or -(CH2CH2O) n -, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0009] Preferably, the E is selected from the following fragments:

[0010]

[0011] Another object of the present invention is to provide a method for preparing the streptavidin containing a long-chain mercapto arm, which comprises the following steps:

[0012] 1) Dissolve the cysteine-type recombinant core streptavidin in an appropriate buffer solution, expose the cysteine residues under the action of an excessive reducing agent, and then separate and remove the excessive reducing agent;

[0013] 2) React the solution obtained in step 1) with an excessive cross-linking agent, and then separate and remove the excessive cross-linking agent;

[0014] 3) Expose the thiol groups in the solution obtained in step 2) under the action of an excessive reducing agent, and then separate and remove the excessive reducing agent to obtain the streptavidin containing a long-chain mercapto arm.

[0015] The appropriate buffer solution is an acetate buffer solution, a phosphate buffer solution or a citrate buffer solution with a pH of 4.0 to 10.0;

[0016] The reducing agent is 2-mercaptoethanol, dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride;

[0017] The cross-linking agent contains a molecule with the structure of formula II:

[0018]

[0019] wherein, E is selected from: a C1-C 10 linear alkyl group, a C3-C 10 branched alkyl group, a C3-C8 cyclic alkyl group, a C6-C 10 aryl group or -(CH2CH2O)n-, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0020] Another object of the present invention is to provide the application of the streptavidin containing a long-chain mercapto arm in enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, time-resolved immunofluorescence assay (TRFIA), quantitative PCR, single-stranded DNA preparation, biomolecule purification, monoclonal antibody preparation.

[0021] Another object of the present invention is to provide the application of the streptavidin containing a long-chain mercapto arm prepared by the above preparation method in enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, time-resolved immunofluorescence assay (TRFIA), quantitative PCR, single-stranded DNA preparation, biomolecule purification, monoclonal antibody preparation.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The streptavidin containing long-chain mercapto arms prepared in this application solves the problem of low bonding efficiency between cysteine-type recombinant core streptavidin and the carrier. Description of the Drawings

[0024] Figure 1 1H NMR spectrum of crosslinking agent A in Example 1.

[0025] Figure 2 Identification result diagram of SDS-PAGE in Example 2.

[0026] Figure 3 Identification result diagram of MALDI-TOF / TOF of streptavidin C with long-chain mercapto arms in Example 2.

[0027] Figure 4 Identification result diagram of MALDI-TOF / TOF of control cysteine-type recombinant core streptavidin in Example 2.

[0028] Figure 5 Identification result diagram of SDS-PAGE in Example 3. Detailed Description of the Invention

[0029] Example 1

[0030] This example provides a preparation method of crosslinking agent A:

[0031] The structural formula of crosslinking agent A is as follows:

[0032]

[0033] Prepared with reference to Journal of Physical Chemistry B, 2004, 108, 16508-16517. The specific preparation process is as follows: In a stirred suspension of cystamine dihydrochloride (230 mg, 1 mmol) and dipropylethylamine (514 μL, 3 mmol) in CH2Cl2 (10 ml), a solution of N-succinimidyl 6-(maleimidocaproate) (310 mg, 1 mmol) in CH2Cl2 (5 ml) was added. After the reaction mixture was stirred for 16 h, the reaction progress was detected by TLC (ethyl acetate). After the reaction was complete, the precipitate was removed by filtration, and the solvent in the filtrate was removed by distillation under reduced pressure to obtain a solid-liquid mixture. Purified by column chromatography to obtain 78 mg of white solid. The identification result of 1H NMR spectrum is as Figure 1 shown, and it can be known from Figure 1 the following: 11H NMR (400 MHz, CDCl3) δ: 6.69 (s, 4H), 6.33 (t, J = 5.4 Hz, 2H), 3.57 (q, J = 6.3 Hz, 4H), 3.51 (t, J = 7.2 Hz, 4H), 2.83 (t, J = 6.4 Hz, 4H), 2.21 (t, J = 7.5 Hz, 4H), 1.71 - 1.57 (m, 8H), 1.36 - 1.28 (m, 4H).

[0034] Example 2

[0035] This example provides the preparation process of streptavidin containing a long-chain mercapto arm:

[0036] (1) Reduction of cysteine-type recombinant core streptavidin: Dissolve 8 mg of cysteine-type recombinant core streptavidin (provided by Hangzhou New Dragon Biotechnology Co., Ltd.) in 1 mL of PBS buffer (pH = 7.4), add 80 μL of a PBS solution of dithiothreitol (59 mg / mL, pH = 7.4), mix by inversion at room temperature for 30 min, then stir and dialyze to remove the excess dithiothreitol, obtaining the reduced cysteine-type recombinant core streptavidin solution.

[0037] (2) Take 0.25 mL of the above-reduced cysteine-type recombinant core streptavidin solution, and add 2, 4, 8, and 16 μL of the DMSO solution (16 mg / mL) of crosslinker A (prepared in Example 1) respectively. Mix by inversion at room temperature for 24 h, then stir and dialyze to remove the excess crosslinker A, obtaining streptavidin conjugate B. Its SDS-PAGE identification is as Figure 2 shown.

[0038] Figure 2 In it, M is the protein molecular weight standard, and lanes 1, 2, 3, and 4 represent the solutions added with 2, 4, 8, and 16 μL of crosslinker A respectively. In lane 4 where 16 μL of crosslinker was added, there are a large number of dimer bands with a molecular weight of about 26 kDa as shown in formula B, proving successful crosslinking. In addition, the band with a molecular weight of about 13 kDa may be the by-product shown in formula B1. Both B and B1 are reduced to streptavidin C containing a long-chain mercapto arm in the subsequent reduction reaction.

[0039]

[0040] (3) Add 20 μL of a PBS solution of dithiothreitol (59 mg / mL, pH = 7.4) to the above streptavidin conjugate B, mix by inversion at room temperature for 30 min, then stir and dialyze to remove the excess dithiothreitol, obtaining streptavidin C containing a long-chain mercapto arm.

[0041]

[0042] The MALDI-TOF / TOF identification results are as follows Figure 3 , 4 as shown. Among them, Figure 3 is streptavidin C with a long-chain mercapto arm, Figure 4 is unmodified cysteine-type recombinant core streptavidin (prepared according to step (1) of this example). It can be seen from Figure 4 that streptavidin exists in the form of dimers, trimers, and tetramers under the conditions of MALDI-TOF / TOF. By comparing the molecular weights of Figure 3 and Figure 4 , it can be known that the long-chain mercapto arm has been successfully attached to streptavidin, and the product quality distribution is uniform.

[0043] Example 3

[0044] This example provides a method for bonding streptavidin with a long-chain mercapto arm to PEG:

[0045] Quantify the protein of streptavidin C with a long-chain mercapto arm prepared in Example 2. Take unmodified cysteine-type recombinant core streptavidin (prepared according to step (1) of Example 2) as a control, and add 0.1 mg of PEG (20 kD) to each mg of cysteine-type recombinant core streptavidin. After inverting and mixing for 1 h, add 3 μL of ethanolamine to each milliliter of the solution, and invert and mix. The SDS-PAGE identification is as follows Figure 5 as shown.

[0046] Figure 5 In it, M is the protein molecular weight standard, lane 1 is the bonding result of unmodified cysteine-type recombinant core streptavidin and PEG, lane 2 is the bonding result of streptavidin C with a long-chain mercapto arm and PEG, and the band near 66 kD represents streptavidin that has not been bonded to PEG. The 66 kD band in lane 2 is significantly less than the 66 kD band in lane 1, indicating that after modification with a long-chain mercapto arm, the bonding efficiency of streptavidin to PEG is significantly improved.

[0047] The above-described examples are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A streptavidin containing a long-chain mercapto arm, characterized in that The structural formula of the streptavidin containing a long-chain mercapto arm is as shown in I.

2. A method for preparing streptavidin containing a long-chain mercapto arm as described in claim 1, characterized in that, The preparation method includes the following steps: 1) Dissolve the cysteine-type recombinant core streptavidin in an appropriate buffer solution. After exposing the cysteine residues under the action of an excessive reducing agent, separate and remove the excessive reducing agent. 2) React the solution obtained in step 1) with an excessive cross-linking agent, and then separate and remove the excessive cross-linking agent. 3) Expose the thiol groups in the solution obtained in step 2) under the action of an excessive reducing agent, and separate and remove the excessive reducing agent to obtain the streptavidin containing a long-chain mercapto arm. The cross-linking agent contains a molecule with the structure of formula II.

3. The preparation method according to claim 2, wherein The appropriate buffer solution is an acetate buffer solution, a phosphate buffer solution or a citrate buffer solution with a pH of 4.0 to 10.

0.

4. The preparation method according to claim 2, wherein The reducing agent is 2-mercaptoethanol, dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride.

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