CN110218237B - Method for coupling negative charge protein with 3-aminopropyl triethoxysilane - Google Patents
Method for coupling negative charge protein with 3-aminopropyl triethoxysilane Download PDFInfo
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Abstract
The invention discloses a method for coupling negative charge protein with 3-aminopropyltriethoxysilane, belonging to the technical field of inorganic materials and biomacromolecule chemistry. The invention controls the pH of the protein activation process and the coupling process of the protein and the 3-aminopropyltriethoxysilane, namely the pH of the activation process is controlled to be 4.5-7.2, the pH of the coupling process is controlled to be 7-8, and the difference between the pH of the solution and the isoelectric point of the protein is ensured to be more than 1. The invention solves the problems of protein coagulation and self-coupling in the process of APTS modification of protein, has high modification efficiency and uniform modification, obviously improves the stability of the protein under non-physiological conditions, and lays a good foundation for preparing nano silicon dioxide encapsulated protein nano materials.
Description
Technical Field
The invention relates to a method for improving protein stability, in particular to a protein amidation modification method for preparing nano silicon dioxide encapsulated protein, which is applied to the technical field of protein modification or composite preparation of organic materials and biomacromolecule chemistry.
Background
Proteins are the material basis of life, and there is no life without proteins. In recent years, due to the diversity of protein functions, more and more protein products are produced and applied to various fields. However, the intrinsic instability of native proteins under non-physiological conditions, such as denaturation, enzymatic digestion, etc., limits their use in industrial or normal laboratory conditions. Therefore, improvement of protein stability is imminent.
Nanosilicon dioxide encapsulated proteins have proven to be an effective method for increasing protein stability. The method is an essential step for encapsulating negatively charged protein by nano silicon dioxide by catalyzing the amino group of 3-Aminopropyltriethoxysilane (APTS) to form an amido bond with the carboxyl group on the protein by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). However, the use of EDC and NHS to catalyze amide bond formation requires adjustment of pH and reaction time. Also proteins are very sensitive to pH. If the pH of the reaction system is not proper in the reaction process, coupling failure such as protein coagulation and self-coupling is likely to be caused, so that subsequent nano-silica cannot complete the encapsulation of the protein. Therefore, pH control in the first protein modification stage in the nanosilicon dioxide encapsulated negatively charged protein method is necessary, and is a technical problem to be solved.
In addition, the carboxyl intermediate after protein activation is greatly affected by pH and reaction time. The highly reactive intermediate can be maintained at pH =7 for 4-5 hours, at pH =8 for 1 hour, and at pH =8.6 for 10 minutes. Proteins are sensitive to pH and often aggregate and denature due to changes in pH. Meanwhile, the protein itself has amino and carboxyl groups, which may cause self-coupling of the protein if the pH of the system is not well controlled.
Disclosure of Invention
In order to solve the problems of the prior art, the invention aims to overcome the defects in the prior art and provide a method for coupling 3-aminopropyltriethoxysilane with negatively charged protein, which respectively considers the difference of the degrees of the activation process and the coupling process influenced by pH, respectively regulates and controls the pH of different mixed liquid systems in the activation process and the coupling process, and effectively controls and prevents the phenomena of protein coagulation and self-coupling in the APTS protein modification process.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for coupling negatively charged proteins to 3-aminopropyltriethoxysilane, comprising the steps of:
a. and (3) an activation stage:
at room temperature conditions, according to protein: EDC: the molar ratio of NHS was 1: (25-100): (50-250) mixing the protein, EDC and NHS in a buffer solution to obtain a mixed solution system containing activated protein, wherein the pH of the mixed solution system is maintained between 4.5-7.2, the difference between the pH of the mixed solution system and the isoelectric point of the protein is at least 1.0, and performing magnetic stirring on the mixed solution system for 15-30min to uniformly disperse the protein in the mixed solution system and prevent the protein from coagulation and self-coupling;
b. a coupling stage:
adding buffer solution and APTS into the mixed solution prepared in the step a to obtain a reaction solution system, adjusting the pH of the reaction solution system by using the buffer solution or acid, and controlling the molar ratio of APTS to protein to be mixed to be (100-1000): 1, maintaining the pH value of the reaction solution system between 7.0 and 8.0, simultaneously enabling the pH value of the reaction solution system to be different from the isoelectric point of the protein by at least 1.0, carrying out magnetic stirring on the mixed solution system for 2 to 12 hours to generate a coupling reaction, modifying the protein by APTS, and enabling the protein not to generate coagulation; controlling the environmental temperature of the reaction solution system to be 4-25 ℃, and regulating and controlling the environmental temperature of the reaction solution system according to the length of the reaction time; after the coupling reaction is finished, the product of protein coupling APTS is ultrafiltered and washed by buffer solution or ultrapure water by using an ultrafiltration tube, and pure negative charge protein coupling 3-aminopropyltriethoxysilane is obtained.
Preferably, the buffer is phosphate buffer PB or Tris-HCl buffer.
The concentration of the buffer solution is preferably 0.1M and the pH is preferably 6.5 to 7.5.
Preferably, the protein is at least one of bovine serum albumin BSA and Insulin.
The ambient temperature is preferably controlled at room temperature at 25 ℃ or 4 ℃. Preferably, the reaction time is short and controlled at room temperature, and the reaction time is long and controlled at 4 ℃.
The invention combines the condition that the process of EDC and NHS catalytic acylation reaction is greatly influenced by pH, maintains the pH of a Ruyi solution system between 4.5 and 7.2 in the process of protein activation, ensures that the activation efficiency reaches the highest level, and effectively avoids protein coagulation and self-coupling. The pH value of the solution system is maintained between 7 and 8 in the coupling process, the coupling efficiency reaches the highest level, and protein coagulation is effectively avoided. After the coupling reaction is finished, the product of the pure protein coupling APTS can be obtained after ultrafiltration washing for a plurality of times by proper buffer solution or ultrapure water through a proper ultrafiltration tube.
Compared with the prior art, the invention has the following obvious and prominent substantive characteristics and remarkable advantages:
1. the method solves the coagulation and self-coupling phenomena of the protein in the modification process by regulating the pH;
2. the method has high protein modification efficiency and uniform modification, and lays a solid foundation for the subsequent synthesis of nano silicon dioxide encapsulated protein nanoparticles with uniform particles, good dispersibility and high stability.
Drawings
Fig. 1 is a Zeta potential diagram of native BSA at buffer pH = 8.0.
Fig. 2 is a Zeta potential diagram of APTS-modified BSA obtained in example of the present invention at pH =8.0 in the buffer system.
Fig. 3 is a graph of Zeta potential of APTS-modified BSA without pH adjustment during modification in the comparative example at pH =8.0 in the buffer system.
FIG. 4 is a TEM image of a transmission electron microscope for preparing nano-silica-encapsulated BSA nanoparticles by using a protein-coupled APTS product prepared in one embodiment of the present invention.
FIG. 5 is a TEM image of a Transmission Electron Microscope (TEM) for preparing nano-silica-encapsulated BSA nanoparticles by using a protein-coupled APTS product prepared by the method of the embodiment of the invention without adjusting pH during protein modification.
Fig. 6 is a Zeta potential diagram of native insulin at buffer system pH = 8.0.
Fig. 7 is a Zeta potential diagram of APTS-modified insulin obtained in example two of the present invention under the condition of buffer system pH = 8.0.
FIG. 8 is a TEM image of the nano-silica encapsulated insulin nanoparticles prepared by the protein-coupled APTS prepared by the method of the second embodiment of the present invention.
Detailed Description
The above protocol is further illustrated below with reference to specific examples, which use BSA (PI = 5.3) and insulin (PI = 5.4) as examples. The preferred embodiments of the invention are detailed below:
the first embodiment is as follows:
in this example, a method for coupling a negatively charged protein to 3-aminopropyltriethoxysilane, comprises the steps of:
a. an activation stage:
dissolving 15mg BSA in 2ml PB in a 50ml eggplant-shaped bottle at room temperature, wherein the PB has pH =6.5, the concentration of PB is 0.1M, and transferring the BSA buffered mixed solution to a chromatography cabinet at 4 ℃, wherein the rotation speed is 750rpm, so that the BSA buffered mixed solution is uniformly mixed; subsequently adding 4.4mg of EDC and 6.6mg of NHS into a BSA buffer mixed solution in a 50ml eggplant-shaped bottle, and magnetically stirring for 30min to obtain a mixed solution system, maintaining the pH of the mixed solution system between 4.5 and 7.2, and simultaneously making the difference between the pH of the mixed solution system and the isoelectric point of the protein be more than 1.0, so that the protein is uniformly dispersed in the mixed solution system, and the protein is not subjected to coagulation and self-coupling;
b. a coupling stage:
adding 13ml of PB with the pH value of 7.5 and the concentration of 0.1M into the mixed solution prepared in the step a to adjust the pH value of the reaction solution, adding 40 mu l of APTS to obtain a reaction solution system with the pH value of 7.4, stirring the reaction solution at the rotation speed of 1200rpm for 12h, simultaneously enabling the pH value of the reaction solution system to be different from the isoelectric point of the protein by more than 1.0, controlling the environmental temperature of the reaction solution system to be 4 ℃, enabling the reaction solution system to generate coupling reaction, modifying the protein by using the APTS, and enabling the protein not to generate coagulation; and after the coupling reaction is finished, using a 30K ultrafiltration tube and adopting ultrapure water to carry out ultrafiltration washing on a product of the protein coupling APTS so as to obtain pure negative charge protein coupling 3-aminopropyltriethoxysilane. In this example, BSA with PI of 5.3 was used, and EDC and NHS were used to catalyze the acylation reaction in combination with the coupling process to obtain pure APTS-modified BSA, i.e., BSA-APTS.
Analysis of experimental tests
The negatively charged protein prepared in the embodiment is coupled with 3-aminopropyltriethoxysilane to prepare the nano-silica encapsulated BSA nanoparticle BSA @ silica, and the steps are as follows:
weighing 7.06mg of arginine, dissolving in 20ml of ultrapure water, and uniformly stirring to obtain an arginine solution, adding 17ml of arginine solution into a 50ml eggplant-shaped bottle, and adjusting the rotating speed of a magnetic stirrer to 1200rpm. Then, 200. Mu.l of the product BSA-APTS prepared in this example was added to form a mixed solution so that the concentration of BSA-APTS in the mixed solution was 0.01mg/ml. Then 100 mul of tetraethoxysilane TEOS and 80 mul of cyclohexane Cyc are mixed to obtain a TEOS/Cyc solution, and the TEOS: volume ratio of Cyc 5:4. then, the above mixed solution containing BSA-APTS was added dropwise to an ethyl orthosilicate solution in an amount of 6. Mu.l per half hour to obtain a reaction solution, and 18. Mu.l of the ethyl orthosilicate solution was added in total to react at room temperature and 25 ℃ for 12 hours. Then according to the same feeding method, 18 mul TEOS/Cyc solution is continuously added after 12 hours, thereby obtaining the BSA nanoparticle BSA @ silica with the particle size of 15 nm.
The Zeta potential of APTS-modified BSA obtained in this example under buffer pH =8.0 is shown in fig. 2. FIG. 4 is a TEM image of a transmission electron microscope for preparing nano-silica-encapsulated BSA nanoparticles by using a protein-coupled APTS product prepared in one embodiment of the present invention.
Comparative example the Zeta potential of APTS-modified BSA without pH adjustment during modification under the conditions of buffer pH =8.0 is shown in fig. 3. Fig. 1 is a Zeta potential diagram of native BSA at buffer pH = 8.0. FIG. 5 is a TEM image of a Transmission Electron Microscope (TEM) for preparing nano-silica-encapsulated BSA nanoparticles by using a protein-coupled APTS product prepared by the method of the embodiment of the invention without adjusting pH during protein modification.
As can be seen from FIGS. 1 to 5, the coupling efficiency was high while maintaining pH at 7.4 during the coupling. This example effectively controls the pH of the protein activation process and the coupling of the protein to 3-aminopropyltriethoxysilane. The pH value of the activation process is controlled to be 4.5-7.2, the pH value of the coupling process is controlled to be 7.4, and the difference between the pH value of the solution and the isoelectric point of the protein is ensured to be more than 1. The method solves the problems of protein coagulation and self-coupling in the process of modifying the protein by APTS, and the invention has high modification efficiency and uniform modification. By adjusting the pH value, the pH values of the APTS modified BSA in the buffer system obtained in this example and the natural BSA in the comparative example were both 8.0, and it was found that pure APTS modified BSA, i.e., BSA-APTS, could be obtained by the method of this example. The product of protein coupling APTS prepared by the method of the embodiment of the invention is used for preparing the BSA nano-particles with uniform particles and good dispersibility, and the product of protein coupling APTS prepared by the method of the embodiment of the invention is used for preparing the BSA nano-particles with poor dispersibility and obvious agglomeration phenomenon under the condition of not adjusting pH in the process of modifying protein.
Example two:
this embodiment is substantially the same as the first embodiment, and is characterized in that:
in this embodiment, a method for coupling 3-aminopropyltriethoxysilane to a negatively charged protein comprises the steps of:
a. and (3) an activation stage:
at room temperature, 5mg of Insulin Insulin was weighed out and dissolved in 1.8ml of Tris-HCl buffer, pH 7.4, at a concentration of 0.1M, and 200. Mu.l of hydrochloric acid at a concentration of 0.12M was added. Then transferring 2ml of the dissolved mixed solution of the Insulin into a 50ml eggplant-shaped bottle, and adjusting the rotating speed of a magnetic stirrer to 700rpm to uniformly mix the mixed solution of the Insulin; subsequently, 6ml of Tris-HCl buffer solution with pH 7.4 and concentration 0.1M was added to adjust the pH. Adding 4.4mg of EDC and 6.6mg of NHS into the mixed solution of Insulin in a 50ml eggplant-shaped bottle, magnetically stirring for 30min to obtain a mixed solution system, maintaining the pH of the mixed solution system to be 6.5, and simultaneously enabling the difference between the pH of the mixed solution system and the isoelectric point of protein to be more than 1.0, so that the protein is uniformly dispersed in the mixed solution system, and the protein is prevented from coagulation and self-coupling;
b. a coupling stage:
adding 13ml of PB with the pH value of 7.5 and the concentration of 0.1M into the mixed solution prepared in the step a to adjust the pH value of the reaction solution, adding 20 mul of APTS to obtain a reaction solution system with the pH value of 7.2, stirring the reaction solution at the rotating speed of 700rpm for 2h, simultaneously enabling the pH value of the reaction solution system to be different from the isoelectric point of the protein by more than 1.0, controlling the environmental temperature of the reaction solution system to be 25 ℃, enabling the reaction solution system to generate coupling reaction, modifying the protein by using the APTS, and enabling the protein not to generate coagulation; after the coupling reaction is finished, a 30K ultrafiltration tube is used, and a buffer solution with pH of 8.0 and concentration of 20mM is adopted to carry out ultrafiltration washing on a product of protein coupling APTS, so that pure negative charge protein coupling 3-aminopropyltriethoxysilane is obtained. In this example, pure Insulin with PI of 5.4 was obtained by EDC and NHS catalyzed acylation in combination with coupling to obtain pure functionalized Insulin with APTS, i.e. Insulin-APTS.
Analysis of experimental tests
The method for preparing the nanometer silica-encapsulated Insulin nano particle Insulin @ silica by using the negative charge protein prepared in the embodiment to couple with 3-aminopropyltriethoxysilane comprises the following steps:
weighing 7.06mg of arginine Arg, dissolving in 20ml of ultrapure water, uniformly stirring to obtain an arginine solution, adding 17ml of arginine solution into a 50ml eggplant-shaped bottle, and adjusting the rotating speed of a magnetic stirrer to 1200rpm. 200 μ l of the product Insulin-APTS prepared in this example were then added to form a mixed solution such that the concentration of Insulin-APTS in the mixed solution was 0.015mg/ml. Then 100 mul tetraethyl orthosilicate TEOS is mixed with 80 mul cyclohexane Cyc to obtain TEOS/Cyc solution, and the TEOS: volume ratio of Cyc 5:4. then, the solution of tetraethoxysilane was added dropwise to the above mixed solution containing Insulin-APTS at a rate of 6. Mu.l per half hour to obtain a reaction solution, and 18. Mu.l of tetraethoxysilane solution was added in total to react at room temperature and 25 ℃ for 12 hours. And then according to the same feeding method, continuously adding 18 mu l of TEOS/Cyc solution after 12h to obtain the nanometer silica packaging Insulin nano particle Insulin @ silica with the particle size of 8 nm. After the reaction is finished, washing the reaction product for several times by using ultrapure water through a 10K ultrafiltration tube to obtain the purer Insulin @ silica.
Fig. 7 is a Zeta potential diagram of APTS modified insulin obtained in example two of the present invention under the condition of pH =8.0 of the buffer system. FIG. 8 is a TEM image of a transmission electron microscope for preparing nano-silica encapsulated insulin nanoparticles by using a product of protein-coupled APTS prepared by the second method in example 8 of the present invention.
Fig. 6 is a Zeta potential diagram of natural insulin under the condition of buffer system pH = 8.0.
As can be seen from FIGS. 6 to 8, the coupling efficiency was high while maintaining pH at 7.2 during the coupling. This example effectively controls the pH of the protein activation process and the coupling process of the protein to 3-aminopropyltriethoxysilane. The pH value of the activation process is controlled to be 6.5, the pH value of the coupling process is controlled to be 7.2, and the difference between the pH value of the solution and the isoelectric point of the protein is ensured to be more than 1. The method solves the problems of protein coagulation and self-coupling in the process of modifying the protein by APTS, and the invention has high modification efficiency and uniform modification. By adjusting the pH value, the pH value of the APTS modified Insulin obtained in this example in the buffer system and the pH value of the natural Insulin obtained in the comparative example in the buffer system are both 8.0, and it can be seen that the method of this example can obtain a pure APTS modified Insulin, i.e., insulin-APTS. The nanometer silica-encapsulated Insulin nanoparticles prepared by the protein coupling APTS product prepared by the method of the embodiment two are uniform in particle size and good in dispersibility, and the nanometer silica-encapsulated Insulin nanoparticles prepared by the protein coupling APTS product prepared by the method of the embodiment two are poor in dispersibility and form obvious agglomeration phenomenon under the condition that the pH is not adjusted in the protein modification process.
As can be seen from the above-mentioned examples of the present invention, the ambient temperature of the reaction system is controlled to be 25 ℃ or 4 ℃. The reaction time is controlled at room temperature when the reaction time is short, the reaction time is controlled at 4 ℃ when the reaction time is long, the reaction conditions are met, the difference of the degrees of the activation process and the coupling process influenced by pH is fully considered, the pH of different mixed liquid systems is respectively regulated and controlled in the activation process and the coupling process, and the phenomena of protein coagulation and self-coupling are effectively controlled and prevented in the process of modifying the protein by APTS.
While the present invention has been described with reference to the above embodiments, the present invention is not limited to the above embodiments, and various changes, modifications, substitutions, combinations or simplifications made according to the spirit and principle of the present invention should be made in an equivalent manner without departing from the technical principle and inventive concept of the method for coupling 3-aminopropyltriethoxysilane to a negatively charged protein.
Claims (3)
1. A method for coupling 3-aminopropyltriethoxysilane to a negatively charged protein, comprising the steps of:
a. an activation stage:
at room temperature conditions, according to protein: EDC: the molar ratio of NHS was 1: (25-100): (50-250) mixing the protein, EDC and NHS in a buffer solution to obtain a mixed solution system containing activated protein, wherein the pH of the mixed solution system is maintained between 4.5-7.2, the difference between the pH of the mixed solution system and the isoelectric point of the protein is at least 1.0, and performing magnetic stirring on the mixed solution system for 15-30min to uniformly disperse the protein in the mixed solution system and prevent the protein from coagulation and self-coupling; the protein is at least one of bovine serum albumin BSA and Insulin;
b. a coupling stage:
adding buffer solution and APTS into the mixed solution prepared in the step a to obtain a reaction solution system, adjusting the pH of the reaction solution system by using the buffer solution or acid, and controlling the molar ratio of APTS to protein to be mixed to be (100-1000): 1, maintaining the pH value of the reaction solution system between 7.0 and 8.0, simultaneously enabling the pH value of the reaction solution system to be different from the isoelectric point of the protein by at least 1.0, carrying out magnetic stirring on the mixed solution system for 2 to 12 hours to generate a coupling reaction, modifying the protein by APTS, and enabling the protein not to generate coagulation; controlling the environmental temperature of the reaction solution system to be 4-25 ℃, and regulating and controlling the environmental temperature of the reaction solution system according to the length of the reaction time; after the coupling reaction is finished, the product of protein coupling APTS is ultrafiltered and washed by buffer solution or ultrapure water by using an ultrafiltration tube, and pure negative charge protein coupling 3-aminopropyltriethoxysilane is obtained.
2. The method of coupling 3-aminopropyltriethoxysilane to a negatively charged protein of claim 1, wherein: the buffer solution is phosphate buffer solution PB or Tris-HCl buffer solution.
3. The method of coupling 3-aminopropyltriethoxysilane to a negatively charged protein according to claim 2, wherein: the concentration of the buffer solution is 0.1M, and the pH value is 6.5-7.5.
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