Preparation method of protein multilayer film

Abstract

The invention relates to the technical field of protein assembly, in particular to a preparation method of a protein multilayer film, which comprises the following steps: immersing a pretreated base material into a poly (allylamine hydrochloride) aqueous solution, taking out and drying, immersing into a mercapto acid aqueous solution, taking out and drying, and washing to obtain a prefabricated base material; then alternately immersing into a protein A solution and a protein B solution, carrying out a layer-by-layer self-assembly reaction by utilizing the protein A and the protein B, and repeating the alternate immersion operation to obtain a protein multilayer film; wherein the A protein is SpyTag-ELP-cuta-ELP-SpyTag, and the amino acid sequence of the A protein is as shown in SEQ ID NO. 1; the protein A is SpyCatcher-ELP-cuta-ELP-SpyCatcher, the protein B is SpyCatcher-ELP-cuta-ELP-SpyCatcher, and the amino acid sequence of the protein B is as shown in SEQ ID NO. 2; the multilayer film structure obtained by the invention has better orderliness and better film stability.

Description

Technical Field

[0001] The invention relates to the technical field of protein assembly, and in particular to a method for preparing a protein multilayer film. Background Art

[0002] It is an effective way to prepare bioactive composite materials by directional assembly of protein molecules into high-order structures. Layer-by-layer (LbL) self-assembly is a versatile surface / interface engineering technology that uses weak interactions between molecules (such as electrostatic effects, hydrogen bonds, coordination bonds, etc.) to form an assembled multilayer film system, and can prepare multilayer film structures with a certain thickness and composition. At present, there are many self-assembly methods, such as immersion, spin coating, spraying, electrodeposition, etc., and the research focus of layer-by-layer self-assembly is mainly on the innovation of self-assembly materials and applications. Different self-assembly materials and methods can directly regulate the physical and chemical properties of the film, such as thickness, uniformity, and interlayer interactions.

[0003] In the prior art, polymer materials are usually used to prepare multilayer membrane structures, but there are also a few works that use proteins to prepare protein-based layer-by-layer self-assembled multilayer membranes. For example, Chinese patent CN106798947A discloses a method for preparing a self-assembled protein multilayer membrane, which uses the hydrogen bonding between proteins and plant polyphenols for layer-by-layer self-assembly. Proteins such as lysozyme, etc., can be seen from their QCM images that the layer-by-layer assembled multilayer membrane structure has poor orderliness, resulting in poor stability, which will have an adverse effect on its application effect. Summary of the invention

[0004] In order to solve the technical problem of poor orderliness of the layer-by-layer self-assembled multilayer membrane structure in the prior art, a method for preparing a protein multilayer membrane is provided. The present invention uses a method of a mutually orthogonal protein chemical reaction pair of SpyTag (represented by A in the following protein A) and SpyCatcher (represented by B in the following protein B) to obtain a protein multilayer membrane structure. The multilayer membrane structure obtained by the present invention has good orderliness and good film stability.

[0005] In order to achieve the above objectives, the present invention is implemented by the following technical solutions:

[0006] The method for preparing a protein multilayer film comprises the following steps:

[0007] S1, immersing the pretreated substrate in a poly(propyleneamine hydrochloride) aqueous solution, taking it out and drying it, then immersing it in a mercapto acid aqueous solution, taking it out and drying it, then rinsing it with a PBS buffer solution to obtain a prefabricated substrate;

[0008] S2, immersing the prefabricated substrate in a protein A solution, taking it out and rinsing it with a PBS buffer solution, and then immersing it in a protein B solution, taking it out and rinsing it with a PBS buffer solution, and performing a layer-by-layer self-assembly reaction using the protein A and the protein B;

[0009] S3. Repeat operation S2 for multiple times to obtain a protein multilayer membrane.

[0010] Furthermore, the protein A in the protein A solution in S2 is SpyTag-ELP-cuta-ELP-SpyTag (represented by AcutaA), and the amino acid sequence of the protein A is as shown in SEQ ID NO.1, consisting of 314 amino acids and a molecular weight of 31653 Daltons.

[0011] Furthermore, the preparation method of the protein A solution is: obtain the target gene fragment SpyTag-ELP-cuta-ELP-SpyTag by 4-step PCR amplification, then introduce the target gene SpyTag-ELP-cuta-ELP-SpyTag into an expression vector to construct a plasmid, express the plasmid in cells, and purify the expressed protein. Preferably, the specific preparation method includes the following process:

[0012] (1) Expression of target gene of protein building block:

[0013] Method for constructing plasmid SpyTag-ELP-cuta-ELP-SpyTag: The target gene fragment was obtained by 4-step PCR amplification: the first step of PCR was to use the existing plasmid (containing SpyTag and ELP sequences) in the laboratory as a template, and primers SpyTag-ELP-F and SpyTag-ELP-R were added to perform PCR amplification to obtain the SpyTag-ELP gene fragment (Table 1); the second step of PCR was to use the existing plasmid (containing ELP and cuta sequences) in the laboratory as a template, and primers ELP-cuta-ELP-F, ELP-cuta-ELP-R were added to perform PCR amplification to obtain the SpyTag-ELP gene fragment (Table 1). P-R is used for PCR amplification to obtain the ELP-cuta-ELP gene fragment; the third step is to use the existing plasmid (containing ELP and SpyTag sequences) in the laboratory as a template, add primers ELP-SpyTag-F and ELP-SpyTag-R to perform PCR amplification to obtain the ELP-SpyTag gene fragment; the fourth step is to perform overlap extension PCR, add the above-obtained SpyTag-ELP, ELP-cuta-ELP, and ELP-SpyTag gene fragments to obtain the target gene SpyTag-ELP-cuta-ELP-SpyTag;

[0014] The amino acid sequence of SpyTag-ELP-cuta-ELP-SpyTag was constructed in the pQE-80L expression vector to obtain a plasmid, which was then transformed into BL21 (DE3) Escherichia coli competent cells and evenly spread on an agar plate containing 100 μg / mL ampicillin. After overnight culture at 37°C, a single clone was selected and inoculated into 6 mL of 2xYT liquid culture medium containing 100 μg / mL ampicillin, and cultured in a shaker at 37°C and 220 rpm for 12 h. The cultured bacterial liquid was inoculated into 200 mL of 2xYT culture medium containing the same concentration of antibiotics and cultured at 37°C until OD 600 =0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, then transfer to 22°C for expression for 12 h and collect the cells;

[0015] (2) Purification of protein building blocks:

[0016] After the expression, the cells were collected by centrifugation at 4°C and 5000g for 15 min. The supernatant was discarded and resuspended with Lysis buffer (50mM disodium hydrogen phosphate, 300mM sodium chloride, 10mM imidazole, pH=8.0) to obtain a resuspension. Phenylmethylsulfonyl fluoride (PMSF) with a final concentration of 1mM was added to the resuspension, and the cells were ultrasonically disrupted in an ice-water bath using an ultrasonic cell disruptor (working for 5s, interval for 8s, power of 260W) to obtain a cell lysate. The cell lysate was centrifuged at 4°C and 28000g for 40min, the supernatant was collected, nickel affinity resin (Ni-NTA) was added to the supernatant, and the cells were mixed on a rotator at 4°C for 1h. After the histidine tag in the protein was fully bound to the resin, it was loaded into an empty column, and the lysate was allowed to flow through. The impurities were eluted with 8-10 times Wash buffer (50mM disodium hydrogen phosphate, 300mM sodium chloride, 20mM imidazole, pH=8.0), and then the cells were washed with 1% elution buffer. The target protein was eluted with PBS (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 250 mM imidazole, pH = 8.0), and finally the target protein was dialyzed into PBS (pH = 7.4) buffer solution to obtain a protein alpha solution with a concentration of 0.5 mg / mL.

[0017] Furthermore, the protein B in the protein B solution in S2 is SpyCatcher-ELP-cuta-ELP-SpyCatcher (represented by BcutaB), and the amino acid sequence of the protein B is as shown in SEQ ID NO.2, consisting of 558 amino acids and a molecular weight of 57808 Daltons.

[0018] Furthermore, the preparation method of the protein B solution is: obtain the target gene fragment SpyCatcher-ELP-cuta-ELP-SpyCatcher by 4-step PCR amplification, then introduce the target gene SpyCatcher-ELP-cuta-ELP-SpyCatcher into an expression vector to construct a plasmid, express the plasmid in cells, and purify the expressed protein. Preferably, the specific preparation method includes the following process:

[0019] (1) Expression of target gene of protein building block:

[0020] Method for constructing plasmid SpyCatcher-ELP-cuta-ELP-SpyCatcher:

[0021] The target gene fragment was obtained by 4-step PCR amplification: the first step was to use the existing plasmid (containing SpyCatcher and ELP sequences) in the laboratory as a template, and add primers SpyCatcher-ELP-F and SpyCatcher-ELP-R for PCR amplification to obtain the SpyCatcher-ELP gene fragment; the second step was to use the existing plasmid (containing ELP and cuta sequences) in the laboratory as a template, and add primers ELP-cuta-ELP-F and ELP-cuta-ELP-R for PCR amplification to obtain the ELP-cuta-ELP gene fragment segment; the third step is to use the existing plasmid (containing ELP and SpyCatcher sequences) in the laboratory as a template, add primers ELP-SpyCatcher-F and ELP-SpyCatcher-R to perform PCR amplification to obtain the ELP-SpyCatcher gene fragment; the fourth step is to perform overlap extension PCR, add the above-obtained SpyCatcher-ELP, ELP-cuta-ELP, and ELP-SpyCatcher gene fragments to obtain the target gene SpyCatcher-ELP-cuta-ELP-SpyCatcher;

[0022] The SpyCatcher-ELP-cuta-ELP-SpyCatcher amino acid sequence was constructed in the pQE-80L expression vector to obtain a plasmid, which was then transformed into BL21 (DE3) Escherichia coli competent cells and evenly spread on an agar plate containing 100 μg / mL ampicillin. After overnight culture at 37°C, a single clone was selected and inoculated into 6 mL of 2xYT liquid culture medium containing 100 μg / mL ampicillin, and cultured in a shaker at 37°C and 220 rpm for 12 h. The cultured bacterial liquid was inoculated into 200 mL of 2xYT culture medium containing the same concentration of antibiotics and cultured at 37°C until OD 600=0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, then transfer to 22°C for expression for 12 h and collect the cells;

[0023] (2) Purification of protein building blocks:

[0024] After the expression, the cells were collected by centrifugation at 4°C and 5000g for 15 min. The supernatant was discarded and resuspended with Lysis buffer (50mM disodium hydrogen phosphate, 300mM sodium chloride, 10mM imidazole, pH=8.0) to obtain a resuspension. Phenylmethylsulfonyl fluoride (PMSF) with a final concentration of 1mM was added to the resuspension, and the cells were ultrasonically disrupted in an ice-water bath using an ultrasonic cell disruptor (working for 5s, interval for 8s, power of 260W). The cell lysate was centrifuged at 4°C and 28000g for 40min, the supernatant was collected, nickel affinity resin (Ni-NTA) was added to the supernatant, and the cells were mixed on a rotator at 4°C for 1h. After the histidine tag in the protein was fully bound to the resin, it was loaded into an empty column, and the lysate was allowed to flow through. The impurities were eluted with 8-10 times Wash buffer (50mM disodium hydrogen phosphate, 300mM sodium chloride, 20mM imidazole, pH=8.0), and then the cells were washed with 1% elution buffer. The target protein was eluted with PBS (50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 250 mM imidazole, pH = 8.0), and finally the target protein was dialyzed into PBS (pH = 7.4) buffer solution to obtain a protein alpha solution with a concentration of 0.5 mg / mL.

[0025] Furthermore, the substrate in S1 is selected from any one of a gold substrate, a quartz substrate, and a silicon dioxide substrate; the pretreatment method of the substrate is: placing the substrate in an ethanol solution (the volume ratio of ethanol to water is 3:7) for ultrasonic treatment, and then immersing it in a mixed solution composed of water, ammonia water (25wt%), and hydrogen peroxide solution (30wt%) in a volume ratio of 5:1:1, soaking at 75°C for at least 30 minutes, and then taking it out and rinsing it with water until the washing solution is neutral.

[0026] Furthermore, the concentration of the poly(propyleneamine hydrochloride) aqueous solution in S1 is 0.1 g / L; the concentration of the thiol acid aqueous solution is 1 mg / mL, and the thiol acid in the thiol acid solution is selected from one or more of thioglycolic acid, thiopropionic acid, normal or isomeric butyric acid containing a thiol group, mercaptosuccinic acid, normal or isomeric valeric acid containing a thiol group, normal or isomeric octanoic acid containing a thiol group, and normal or isomeric hexanoic acid containing a thiol group.

[0027] Further, the reaction temperature of immersing the poly(propyleneamine hydrochloride) aqueous solution in S1 is 25° C., the reaction time is 10 min, and after taking out, it is blown dry with N2; the reaction temperature of immersing the mercapto acid aqueous solution is 25° C., the reaction time is 10 min, and after taking out, it is blown dry with N2;

[0028] The drying temperature in S1 is 100°C and the drying time is 1h;

[0029] The reaction temperature of S2 immersed in the protein A solution is 25° C. and the immersion time is 10-15 min; the reaction temperature of S2 immersed in the protein B solution is 25° C. and the immersion time is 10-15 min.

[0030] Furthermore, the number of repeated operations in S3 is at least 5 times.

[0031] Beneficial technical effects:

[0032] In the process of preparing the multilayer film of the present invention, the substrate is first immersed in a poly(propyleneamine hydrochloride) aqueous solution and a mercapto acid aqueous solution in sequence, so that the two react with amino groups and carboxyl groups to form peptide bonds, thereby achieving adhesion of subsequent specific proteins, so as to smoothly obtain a layer-by-layer self-assembled multilayer film structure;

[0033] The heads and tails of the protein A and protein B used in the present invention have at least three pairs of reactive functional groups, and the middle cuta part is a trimer. The functional groups of the two can sustainably produce layer-by-layer self-assembly reactions to form a multilayer film; the self-assembled multilayer film formed by the two proteins of the middle trimer used in the present invention has good stacking order, thereby showing good film stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the size exclusion chromatography elution curve (SEC) of protein acetabulum;

[0035] Figure 2 is the size exclusion chromatography elution curve (SEC) of protein B;

[0036] Figure 3 This is the SDS-PAGE electrophoresis diagram of protein A and protein B;

[0037] Figure 4 This is a layer-by-layer assembly data diagram of the multilayer film obtained in Example 1 under quartz microcrystal balance detection. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention will be described clearly and completely below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. All other embodiments obtained by ordinary technicians in this field without creative work based on the embodiments of the present invention are within the scope of protection of the present invention.

[0039] Unless otherwise specifically stated, the numerical value set forth in these embodiments does not limit the scope of the present invention. The technology and method known to those of ordinary skill in the relevant art may not be discussed in detail, but in appropriate cases, the technology and method should be considered as a part of the specification. In all examples shown and discussed here, any specific value should be interpreted as being merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments may have different values.

[0040] In addition, it should be noted that the use of words such as "A" and "B" to limit the protein name is only to facilitate the distinction between the proteins used in each reaction step. Unless otherwise stated, the above words have no special meaning and therefore cannot be understood as limiting the scope of protection of the present invention.

[0041] The experimental methods in the following examples without specifying specific conditions are usually measured in accordance with national standards; if there are no corresponding national standards, they are carried out in accordance with the general international standards or the standard requirements proposed by relevant enterprises.

[0042] All the following experiments used Mg-free 2+ ,Ca 2+ PBS buffer solution, and the pH value of the PBS buffer solution is 7.4, and its components are 10mM Na2HPO4, 2mM KH2PO4, 2.7mM KCl, and 137mM NaCl.

[0043] Preparation Example 1

[0044] SpyTag-ELP-cuta-ELP-SpyTag is a fusion protein that combines the properties of the SpyTag or SpyCatcher system with the heat-responsive protein ELP (elastin-like polypeptide). The basic steps to construct this fusion protein are as follows:

[0045] Select SpyTag: SpyTag is a short peptide that can covalently bind to the SpyCatcher protein and is more suitable for insertion into other proteins. The SpyTag and SpyCatcher gene sequences are from Liu, Y. et al. Chem. Sci., 2017, 8, 6577-6582.

[0046] Designing ELP sequence: ELP (elastin-like polypeptide) is a temperature-responsive protein. The phase transition temperature of ELP is determined by the length and amino acid composition of its repeat sequence. Usually, the repeat sequence of ELP consists of a unit repeat of (VPGXG), where X can be any amino acid except proline. Increasing the number of ELP repeats will lead to an increase in the phase transition temperature, while changing the amino acid type of X will affect the phase transition temperature and protein stability. Therefore, by adjusting the number of ELP repeats and the amino acid type of X, a protein that can be reversibly dissolved and precipitated at different temperatures can be designed. The ELP gene sequence comes from Wang, X. et al. Angew. Chem. Int. Ed. 2016, 55, 3442-3446.

[0047] Insert cuta sequence: cuta is a copper divalent cation tolerance protein, a homologous analog of Escherichia coli CutA (coppertolerance A), with a stable trimer structure. The cuta protein used in this experiment is one of its mutant structures (PDB: 2E66).

[0048] Protein sequence design: Combine the sequences of SpyTag, ELP and cuta.

[0049] Gene synthesis: synthesize the corresponding gene according to the designed protein sequence.

[0050] Expression and purification: The synthesized gene is inserted into an expression vector and expressed in an appropriate host cell. After expression, the fusion protein is purified by nickel column affinity chromatography.

[0051] The steps for constructing the SpyCatcher-ELP-cuta-ELP-SpyCatcher fusion protein are similar to those for SpyTag-ELP-cuta-ELP-SpyTag, but SpyCatcher is used instead of SpyTag. SpyCatcher is a longer protein fragment that can covalently bind to SpyTag.

[0052] The specific gene synthesis, expression and purification of SpyTag-ELP-cuta-ELP-SpyTag and SpyCatcher-ELP-cuta-ELP-SpyCatcher are as follows in Examples 1 and 2.

[0053] Table 1 Primer gene sequences for constructing SpyTag-ELP-cuta-ELP-SpyTag plasmid

[0054]

[0055]

[0056] Table 2 Primer gene sequences for constructing SpyCatcher-ELP-cuta-ELP-SpyCatcher plasmid

[0057]

[0058] Example 1

[0059] A protein A solution is prepared, wherein the protein A solution is SpyTag-ELP-cuta-ELP-SpyTag (AcutaA), the amino acid sequence of which is as shown in SEQ ID NO. 1, consisting of 314 amino acids and a molecular weight of 31653 Daltons;

[0060] The preparation method of the protein acetabulum solution is summarized as follows: introducing SpyTag-ELP-cuta-ELP-SpyTag into an expression vector to obtain a plasmid, expressing the plasmid in cells, and purifying the expressed protein.

[0061] The specific steps include:

[0062] (1) Expression of target gene of protein building block:

[0063] The method for constructing SpyTag-ELP-cuta-ELP-SpyTag plasmid is as follows: the target gene fragment is obtained by 4-step PCR amplification: the first step of PCR is to use the existing plasmid (containing SpyTag and ELP sequences) in the laboratory as a template, and add primers SpyTag-ELP-F and SpyTag-ELP-R for PCR amplification to obtain the SpyTag-ELP gene fragment; the second step of PCR is to use the existing plasmid (containing ELP and cuta sequences) in the laboratory as a template, and add primers ELP-cuta-ELP-F and ELP-cuta-ELP-R for PCR amplification to obtain ELP-cuta-ELP P gene fragment; the third step is to use the existing plasmid (containing ELP and SpyTag sequences) in the laboratory as a template, add primers ELP-SpyTag-F and ELP-SpyTag-R to perform PCR amplification to obtain the ELP-SpyTag gene fragment; the fourth step is to perform overlap extension PCR, add the above-obtained SpyTag-ELP, ELP-cuta-ELP, and ELP-SpyTag gene fragments to obtain the target gene SpyTag-ELP-cuta-ELP-SpyTag; PCR amplification is a conventional technical means, and those skilled in the art can synthesize the target gene according to the selection of raw materials;

[0064] The target gene SpyTag-ELP-cuta-ELP-SpyTag amino acid sequence was constructed in the pQE-80L expression vector to obtain a plasmid, which was then transformed into BL21 (DE3) Escherichia coli competent cells and evenly spread on an agar plate containing 100 μg / mL ampicillin. After overnight culture at 37°C, a single clone was selected and inoculated into 6 mL of 2xYT liquid culture medium containing 100 μg / mL ampicillin, and cultured in a shaker at 37°C and 220 rpm for 12 h. The cultured bacterial liquid was inoculated into 200 mL of 2xYT culture medium containing the same concentration of antibiotics and cultured at 37°C until OD 600 =0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, then transfer to 22°C for expression for 12 h and collect the bacteria.

[0065] (2) Purification of protein building blocks:

[0066] After the expression, the cells were collected by centrifugation at 4°C and 5000g for 15 min. The supernatant was discarded and resuspended with Lysis buffer (50mM disodium hydrogen phosphate, 300mM sodium chloride, 10mM imidazole, pH=8.0) to obtain a resuspension. Phenylmethylsulfonyl fluoride (PMSF) with a final concentration of 1mM was added to the resuspension, and the cells were ultrasonically disrupted in an ice-water bath using an ultrasonic cell disruptor (working for 5s, interval for 8s, power of 260W) to obtain a cell lysate. The cell lysate was centrifuged at 4°C and 28000g for 40min, the supernatant was collected, nickel affinity resin (Ni-NTA) was added to the supernatant, and the cells were mixed on a rotator at 4°C for 1h. After the histidine tag in the protein was fully bound to the resin, it was loaded into an empty column, and the lysate was allowed to flow through. The impurities were eluted with 8-10 times Wash buffer (50mM disodium hydrogen phosphate, 300mM sodium chloride, 20mM imidazole, pH=8.0), and then the cells were washed with 1% elution buffer. The target protein was eluted with buffer (50 mM disodium hydrogen phosphate, 300 mM sodium chloride, 250 mM imidazole, pH = 8.0), and finally dialyzed into PBS (pH = 7.4) buffer solution to obtain a protein alpha solution with a concentration of 0.5 mg / mL. The protein alpha in the obtained protein alpha solution was further purified by SEC. The size exclusion chromatography elution curve is shown in Figure 1 As shown, it can be observed from the elution peak that a relatively pure protein acetone product is obtained.

[0067] Example 2

[0068] A protein B solution was prepared, wherein the protein B contained therein was SpyCatcher-ELP-cuta-ELP-SpyCatcher (BcutaB), whose amino acid sequence was as shown in SEQ ID NO.2, consisting of 558 amino acids in total and having a molecular weight of 57808 Daltons.

[0069] The preparation method of protein B solution is summarized as follows: introducing SpyCatcher-ELP-cuta-ELP-SpyCatcher into an expression vector to obtain a plasmid, expressing the plasmid in cells, and purifying the expressed protein.

[0070] The specific steps include:

[0071] (1) Expression of target gene of protein building block:

[0072] Method for constructing SpyCatcher-ELP-cuta-ELP-SpyCatcher plasmid: The target gene fragment was obtained by 4-step PCR amplification: the first step of PCR was to use the existing plasmid (containing SpyCatcher and ELP sequences) in the laboratory as a template, and add primers SpyCatcher-ELP-F and SpyCatcher-ELP-R for PCR amplification to obtain the SpyCatcher-ELP gene fragment (Table 2); the second step of PCR was to use the existing plasmid (containing ELP and cuta sequences) in the laboratory as a template, and add primers ELP-cuta-ELP-F and ELP-cuta-ELP-R for PCR amplification to obtain the ELP-cuta-ELP gene fragment. The third step is to use the existing plasmid (containing ELP and SpyCatcher sequences) in the laboratory as a template, add primers ELP-SpyCatcher-F and ELP-SpyCatcher-R to perform PCR amplification to obtain the ELP-SpyCatcher gene fragment; the fourth step is to perform overlap extension PCR, add the above-obtained SpyCatcher-ELP, ELP-cuta-ELP, and ELP-SpyCatcher gene fragments to obtain the target gene SpyCatcher-ELP-cuta-ELP-SpyCatcher; PCR amplification is a conventional technical means, and those skilled in the art can synthesize the target gene according to the selection of raw materials;

[0073] The SpyCatcher-ELP-cuta-ELP-SpyCatcher amino acid sequence was constructed in the pQE-80L expression vector to obtain a plasmid, which was then transformed into BL21 (DE3) Escherichia coli competent cells and evenly spread on an agar plate containing 100 μg / mL ampicillin. After overnight culture at 37°C, a single clone was selected and inoculated into 6 mL of 2xYT liquid culture medium containing 100 μg / mL ampicillin, and cultured in a shaker at 37°C and 220 rpm for 12 h. The cultured bacterial liquid was inoculated into 200 mL of 2xYT culture medium containing the same concentration of antibiotics and cultured at 37°C until OD 600 =0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, then transfer to 22°C for expression for 12 h and collect the bacteria.

[0074] (2) Purification of protein building blocks:

[0075] After the expression, the cells were collected by centrifugation at 4°C and 5000g for 15 min. The supernatant was discarded and resuspended with Lysis buffer (50mM disodium hydrogen phosphate, 300mM sodium chloride, 10mM imidazole, pH=8.0) to obtain a resuspension. Phenylmethylsulfonyl fluoride (PMSF) with a final concentration of 1mM was added to the resuspension, and the cells were ultrasonically disrupted in an ice-water bath using an ultrasonic cell disruptor (working for 5s, interval for 8s, power of 260W). The cell lysate was centrifuged at 4°C and 28000g for 40min, the supernatant was collected, nickel affinity resin (Ni-NTA) was added to the supernatant, and the cells were mixed on a rotator at 4°C for 1h. After the histidine tag in the protein was fully bound to the resin, it was loaded into an empty column, and the lysate was allowed to flow through. The impurities were eluted with 8-10 times Wash buffer (50mM disodium hydrogen phosphate, 300mM sodium chloride, 20mM imidazole, pH=8.0), and then the cells were washed with 1% elution buffer. The target protein was eluted with buffer (50 mM disodium hydrogen phosphate, 300 mM sodium chloride, 250 mM imidazole, pH = 8.0), and finally dialyzed into PBS (pH = 7.4) buffer solution to obtain a protein A solution with a concentration of 0.5 mg / mL. The obtained protein B was further purified by SEC, and the size exclusion chromatography elution curve was as follows: Figure 2 As shown, pure protein B product can be observed from the elution peak.

[0076] Figure 3The SDS-PAGE electrophoresis diagram of protein A and protein B. The test method is as follows: the sample is mixed with 5 times SDS-PAGE loading buffer (250 mM Tris-HCl, 50 wt% glycerol, 10 wt% SDS, 250 mM β-mercaptoethanol and 0.05 wt% bromophenol blue), heated at 98°C for 10 min, and then Gel exclusion chromatography (SEC) was performed on a Superdex 200 Increase 10 / 300 GL column using PBS (pH = 7.4) as the mobile phase at a flow rate of 0.5 mL / min in a FPLC system (GE Healthcare, Inc.). The specific molecular weight is as follows Figure 3 As shown in the figure, the molecular weights of proteins A and B were verified by SDS-PAGE gel images, which were in line with expectations. It can be seen that there are monomers and trimers in the purified products A and B. Due to the strong non-covalent interaction, the protein denaturation on SDS-PAGE is incomplete, and the migration rate decreases during the gel run. The trimer is located at the front of the gel image.

[0077] Example 3

[0078] The method for preparing a protein multilayer film comprises the following steps:

[0079] S1. Pretreatment of substrate: A gold substrate is selected as the substrate, which is placed in an ethanol solution (the volume ratio of ethanol to water is 3:7) for ultrasonic treatment, and then immersed in a mixed solution composed of water, ammonia water (25wt%), and hydrogen peroxide solution (30wt%) in a volume ratio of 5:1:1, and immersed at 75°C for at least 30 minutes. After being taken out, rinse with water until the washing solution is neutral;

[0080] Then, the pretreated substrate was immersed in a 0.1 g / L poly(propyleneamine hydrochloride) aqueous solution, immersed for 10 min at 25° C., and then dried with N2, and then immersed in a 1 mg / mL thioglycolic acid aqueous solution, immersed for 10 min at 25° C., and then dried with N2, and then rinsed with a PBS buffer solution with a pH of 7.4 to obtain a prefabricated substrate;

[0081] S2, immersing the prefabricated substrate into the 0.5 mg / mL protein A solution prepared in Example 1, taking it out and rinsing it with a PBS buffer solution of pH=7.4, and then immersing it into the 0.5 mg / mL protein B solution prepared in Example 2, taking it out and rinsing it with a PBS buffer solution of pH=7.4, and performing a layer-by-layer self-assembly reaction using protein A and protein B;

[0082] S3. Repeat operation S2 for multiple times to obtain a 22-layer protein multilayer membrane.

[0083] The QCM curve of the multilayer film prepared in this embodiment is as follows: Figure 4 As shown by Figure 4 It can be seen that by alternately adding BcutaB and AcutaA to the AcutaA-4Cys-modified gold surface, a 22-layer protein film can be gradually grown through the covalent coupling of SpyTag / SpyCatcher, demonstrating good LbL assembly efficiency.

[0084] Example 4

[0085] The method for preparing a protein multilayer film comprises the following steps:

[0086] S1. Pretreatment of substrate: A silicon dioxide substrate is selected as the substrate, which is placed in an ethanol solution (the volume ratio of ethanol to water is 3:7) for ultrasonic treatment, and then immersed in a mixed solution composed of water, ammonia water (25wt%), and hydrogen peroxide solution (30wt%) in a volume ratio of 5:1:1, and immersed at 75°C for at least 30 minutes. After being taken out, it is rinsed with water until the washing solution is neutral;

[0087] Then, the pretreated substrate was immersed in a 0.1 g / L poly(propyleneamine hydrochloride) aqueous solution, immersed at 25° C. for 10 min, and then dried with N2, and then immersed in a 1 mg / mL mercaptopropionic acid aqueous solution, immersed at 25° C. for 10 min, and then dried with N2, and then rinsed with a PBS buffer solution to obtain a prefabricated substrate;

[0088] S2, immersing the prefabricated substrate in a 0.5 mg / mL protein A solution, taking it out and rinsing it with a PBS buffer solution of pH=7.4, and then immersing it in a 0.5 mg / mL protein B solution, taking it out and rinsing it with a PBS buffer solution, and performing a layer-by-layer self-assembly reaction using protein A and protein B;

[0089] The protein acetonide in the protein acetonide solution is SpyTag-ELP-cuta-ELP-SpyTag, which is the same as that in Example 1;

[0090] The protein B in the protein B solution is SpyCatcher-ELP-cuta-ELP-SpyCatcher, which is the same as that in Example 2;

[0091] S3. Repeat operation S2 for multiple times to obtain a 22-layer protein multilayer membrane.

[0092] Example 3

[0093] The method for preparing a protein multilayer film comprises the following steps:

[0094] S1. Pretreatment of substrate: A quartz substrate is selected as the substrate, which is placed in an ethanol solution (the volume ratio of ethanol to water is 3:7) for ultrasonic treatment, and then immersed in a mixed solution composed of water, ammonia water (25wt%), and hydrogen peroxide solution (30wt%) in a volume ratio of 5:1:1, and immersed at 75°C for at least 30 minutes. After being taken out, it is rinsed with water until the washing solution is neutral;

[0095] Then, the pretreated substrate was immersed in a 0.1 g / L poly(propyleneamine hydrochloride) aqueous solution at 25°C for 10 min and then dried with N2, then immersed in a 1 mg / mL 4-mercaptobutyric acid aqueous solution at 25°C for 10 min and then dried with N2, and then rinsed with a PBS buffer solution to obtain a prefabricated substrate;

[0096] S2, immersing the prefabricated substrate in a 0.5 mg / mL protein A solution, taking it out and rinsing it with a PBS buffer solution of pH=7.4, and then immersing it in a 0.5 mg / mL protein B solution, taking it out and rinsing it with a PBS buffer solution of pH=7.4, and performing a layer-by-layer self-assembly reaction using protein A and protein B;

[0097] The protein acetonide in the protein acetonide solution is SpyTag-ELP-cuta-ELP-SpyTag, which is the same as that in Example 1;

[0098] The protein B in the protein B solution is SpyCatcher-ELP-cuta-ELP-SpyCatcher, which is the same as that in Example 2;

[0099] S3. Repeat operation S2 for multiple times to obtain a 22-layer protein multilayer membrane.

[0100] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a protein multilayer film, characterized in that: The steps include: S1, immersing the pretreated substrate in a poly(propyleneamine hydrochloride) aqueous solution, taking it out and drying it, then immersing it in a mercapto acid aqueous solution, taking it out and drying it, then rinsing it with a PBS buffer solution to obtain a prefabricated substrate; S2, immersing the prefabricated substrate in a protein A solution, taking it out and rinsing it with a PBS buffer solution, and then immersing it in a protein B solution, taking it out and rinsing it with a PBS buffer solution, and performing a layer-by-layer self-assembly reaction using the protein A and the protein B; The protein A in the protein A solution is SpyTag-ELP-cuta-ELP-SpyTag, and the amino acid sequence of the protein A is as shown in SEQ ID NO.1, consisting of 314 amino acids and having a molecular weight of 31653 Daltons; The protein B in the protein B solution is SpyCatcher-ELP-cuta-ELP-SpyCatcher, and the amino acid sequence of the protein B is as shown in SEQ ID NO.2, consisting of 558 amino acids and a molecular weight of 57808 Daltons; S3. Repeat operation S2 for multiple times to obtain a protein multilayer membrane.

2. The method for preparing a protein multilayer film according to claim 1, characterized in that: The preparation method of the protein A solution is as follows: obtaining the target gene fragment SpyTag-ELP-cuta-ELP-SpyTag through 4-step PCR amplification, then introducing the target gene SpyTag-ELP-cuta-ELP-SpyTag into an expression vector to construct a plasmid, expressing the plasmid in cells, and purifying the expressed protein.

3. The method for preparing a protein multilayer film according to claim 2, characterized in that: The preparation method of the protein acetylcholine solution specifically comprises the following steps: (1) Expression of target gene of protein building block: Method for constructing plasmid SpyTag-ELP-cuta-ELP-SpyTag: The target gene fragment is obtained by 4-step PCR amplification: the first step is to use the plasmid containing SpyTag and ELP sequences already available in the laboratory as a template, add the forward primer and reverse primer of SpyTag-ELP to perform PCR amplification to obtain the SpyTag-ELP gene fragment; the second step is to use the plasmid containing ELP and cuta sequences already available in the laboratory as a template, add the forward primer and reverse primer of ELP-cuta-ELP to perform PCR amplification to obtain the ELP-cuta-ELP gene fragment; the third step is to use the plasmid containing ELP and SpyTag sequences already available in the laboratory as a template, add the forward primer and reverse primer of ELP-SpyTag to perform PCR amplification to obtain the ELP-SpyTag gene fragment; the fourth step is to perform overlap extension PCR, add the SpyTag-ELP, ELP-cuta-ELP, and ELP-SpyTag gene fragments obtained above to obtain the target gene SpyTag-ELP-cuta-ELP-SpyTag; The amino acid sequence of SpyTag-ELP-cuta-ELP-SpyTag was constructed in the pQE-80L expression vector to obtain a plasmid, which was then transformed into BL21 (DE3) Escherichia coli competent cells and evenly spread on an agar plate containing 100 μg / mL ampicillin. After overnight culture at 37°C, a single clone was selected and inoculated into 6 mL of 2xYT liquid culture medium containing 100 μg / mL ampicillin, and cultured in a shaker at 37°C and 220 rpm for 12 h. The cultured bacterial liquid was inoculated into 200 mL of 2xYT culture medium containing the same concentration of antibiotics and cultured at 37°C until OD 600 =0.8, add isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mM, then transfer to 22°C for expression for 12 h and collect the cells; (2) Purification of protein building blocks: After the expression, the cells were collected by centrifugation at 4°C and 5000g for 15 min. The supernatant was discarded and resuspended with Lysis buffer to obtain a resuspension. Phenylmethylsulfonyl fluoride with a final concentration of 1 mM was added to the resuspension, and the cells were ultrasonically disrupted with an ultrasonic cell disruptor in an ice-water bath. The ultrasonic disruption parameters were as follows: working 5s, interval 8s, power 260W, to obtain a cell lysate. The cell lysate was centrifuged at 4°C and 28000g for 40 min, the supernatant was collected, Ni-NTA nickel affinity resin was added to the supernatant, and the cells were mixed on a rotator at 4°C for 1 h. After the histidine tag in the protein was fully combined with the resin, it was loaded into an empty column, and the lysate was waited for to flow through. The impurities were eluted with 8-10 times Wash buffer, and the target protein was eluted with Elution buffer. Finally, the target protein was dialyzed into PBS buffer solution to obtain a protein alpha solution with a concentration of 0.5 mg / mL.

4. The method for preparing a protein multilayer film according to claim 1, characterized in that: The preparation method of the protein B solution is: obtaining the target gene fragment SpyCatcher-ELP-cuta-ELP-SpyCatcher through 4-step PCR amplification, then introducing the target gene SpyCatcher-ELP-cuta-ELP-SpyCatcher into an expression vector to construct a plasmid, expressing the plasmid in cells, and purifying the expressed protein.

5. The method for preparing a protein multilayer film according to claim 4, characterized in that: The preparation method of the protein B solution specifically comprises the following steps: (1) Expression of target gene of protein building block: Method for constructing plasmid SpyCatcher-ELP-cuta-ELP-SpyCatcher: The target gene fragment was obtained by 4-step PCR amplification: the first step was to use the plasmid containing SpyCatcher and ELP sequences already available in the laboratory as a template, add the forward primer and reverse primer of SpyCatcher-ELP to perform PCR amplification to obtain the SpyCatcher-ELP gene fragment; the second step was to use the plasmid containing ELP and cuta sequences already available in the laboratory as a template, add the forward primer and reverse primer of ELP-cuta-ELP to perform PCR amplification to obtain the ELP-cuta-ELP gene fragment; the third step was to use the plasmid containing ELP and SpyCatcher sequences already available in the laboratory as a template, add the forward primer and reverse primer of ELP-SpyCatcher to perform PCR amplification to obtain the ELP-SpyCatcher gene fragment; the fourth step was to perform overlap extension PCR, add the SpyCatcher-ELP, ELP-cuta-ELP, and ELP-SpyCatcher gene fragments obtained above to obtain the target gene SpyCatcher-ELP-cuta-ELP-SpyCatcher; The SpyCatcher-ELP-cuta-ELP-SpyCatcher amino acid sequence was constructed in the pQE-80L expression vector to obtain a plasmid, which was then transformed into BL21 (DE3) Escherichia coli competent cells and then evenly spread on an agar plate containing 100 μg / mL ampicillin. After overnight culture at 37°C, a single clone was selected and inoculated into 6 mL of 2xYT liquid culture medium containing 100 μg / mL ampicillin, and cultured in a shaker at 37°C and 220 rpm for 12 h. The cultured bacterial liquid was inoculated into 200 mL of 2xYT culture medium containing the same concentration of antibiotics and cultured at 37°C until OD 600 =0.8, add isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mM, then transfer to 22°C for expression for 12 h and collect the cells; (2) Purification of protein building blocks: After the expression, the cells were collected by centrifugation at 4°C and 5000g for 15 min. The supernatant was discarded and resuspended with Lysis buffer to obtain a resuspension. Phenylmethylsulfonyl fluoride (PMSF) with a final concentration of 1 mM was added to the resuspension, and ultrasonic cell disruptor was used in an ice-water bath for ultrasonic disruption. The ultrasonic disruption parameters were: working 5s, interval 8s, power 260W, and the cell lysate was centrifuged at 4°C and 28000g for 40min. The supernatant was collected, Ni-NTA nickel affinity resin was added to the supernatant, and mixed on a rotator at 4°C for 1h. After the histidine tag in the protein was fully combined with the resin, it was loaded into an empty column, and the lysate was waited for to flow through. The impurities were eluted with 8-10 times Washbuffer, and the target protein was eluted with Elution buffer. Finally, the target protein was dialyzed into PBS buffer solution to obtain a protein alpha solution with a concentration of 0.5 mg / mL.

6. The method for preparing a protein multilayer film according to any one of claims 1 to 5, characterized in that: The substrate in S1 is selected from any one of a gold substrate, a quartz substrate, and a silicon dioxide substrate; The pretreatment method of the substrate is: placing the substrate in an ethanol solution for ultrasonic treatment, wherein the volume ratio of ethanol to water is 3:7, and then immersing the substrate in a mixed solution composed of water, 25wt% ammonia water, and 30wt% hydrogen peroxide solution in a volume ratio of 5:1:1, immersing at 75°C for at least 30 minutes, and then taking it out and rinsing it with water until the washing solution is neutral.

7. The method for preparing a protein multilayer film according to any one of claims 1 to 5, characterized in that: The concentration of the poly(propyleneamine hydrochloride) aqueous solution in S1 is 0.1 g / L; the concentration of the thiol acid aqueous solution is 1 mg / mL, and the thiol acid in the thiol acid solution is selected from one or more of thioglycolic acid, thiopropionic acid, normal or isomeric butyric acid containing a thiol group, mercaptosuccinic acid, normal or isomeric valeric acid containing a thiol group, normal or isomeric octanoic acid containing a thiol group, and normal or isomeric hexanoic acid containing a thiol group.

8. The method for preparing a protein multilayer film according to any one of claims 1 to 5, characterized in that: The reaction temperature of immersing the poly(propyleneamine hydrochloride) aqueous solution in S1 is 25°C and the reaction time is 10 minutes, and after taking it out, it is blown dry with N2; the reaction temperature of immersing the mercapto acid aqueous solution in S1 is 25°C and the reaction time is 10 minutes, and after taking it out, it is blown dry with N2; The reaction temperature of the protein A solution immersed in S2 is 25°C and the immersion time is 10-15min; the reaction temperature of the protein B solution immersed in S2 is 25°C and the immersion time is 10-15min; The number of repeated operations in S3 is at least 5 times.

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