CN117603953A - Binding protein capable of specifically binding pectin and preparation and application thereof - Google Patents

Binding protein capable of specifically binding pectin and preparation and application thereof Download PDF

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CN117603953A
CN117603953A CN202311572990.1A CN202311572990A CN117603953A CN 117603953 A CN117603953 A CN 117603953A CN 202311572990 A CN202311572990 A CN 202311572990A CN 117603953 A CN117603953 A CN 117603953A
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pectin
reaction medium
binding protein
sample
pecbp
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常耀光
刘冠辰
梅轩玮
薛长湖
李嘉靖
陶雯雯
郑龙
唐庆娟
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Ocean University of China
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Abstract

The invention belongs to the technical field of bioengineering and application thereof, and particularly relates to a binding protein capable of specifically binding pectin, and preparation and application thereof. The amino acid sequence of the specific binding protein is shown as SEQ ID NO.1, and the specific binding protein is prepared by a cloning expression method, can bind pectin, is not bound with other carbohydrates, has the specific binding capacity, and can be used for detecting and semi-quantitatively analyzing pectin.

Description

Binding protein capable of specifically binding pectin and preparation and application thereof
Technical field:
the invention belongs to the technical field of bioengineering and application thereof, and particularly relates to a binding protein capable of specifically binding pectin, and preparation and application thereof.
The background technology is as follows:
pectin is a cell wall component of all higher plants and plays an important role in maintaining cell structure. Pectin is mainly an acidic heteropolysaccharide composed of D-galacturonic acid (Galacturonic acid, galA) linked by alpha-1, 4-glycosidic bond, and is mainly classified into 3 types, namely Homogalacturonan (HG), type I rhamnogalacturonan (type I Rhamngalacturonan, RG-I) and type II rhamnogalacturonan (type II Rhamngalacturonan, RG-II) according to the difference of molecular main chain and branched chain structures. Pectin has various biological activities such as antioxidation, bacteriostasis, anti-inflammatory, immunoregulation, blood sugar reduction, gastrointestinal health regulation and the like, and has functions of gel, emulsification, stabilization, thickening and the like. As a natural food additive, pectin is widely applied to the fields of food, medicine, daily chemicals, textiles and the like, and has market potential.
Carbohydrate-specific binding proteins are a class of non-catalytically active domains with independent structure and function that are capable of specifically recognizing and binding to carbohydrates of specific structure, usually as accessory elements from carbohydrate-active enzymes in bacteria. Antibodies are a common type of binding protein, while saccharides are weak in immunogenicity, and the difficulty of preparing antibodies by animal immunization is high. The carbohydrate specific binding protein widely exists in microorganisms, has clear gene background, can be prepared in batches by cloning and expressing, and has the advantages of small molecular size, high efficiency in acquisition, flexible marking and the like. In recent years, carbohydrate-specific binding proteins are widely used in the fields of enzyme modification, biological materials, molecular probes and the like, and are important tools for carbohydrate-specific detection and in-situ analysis. With the discovery of novel carbohydrate-active enzymes, novel carbohydrate-specific binding proteins have been continuously discovered. There are still a large number of novel sequence, unknown functional domains present in enzymes that have not been resolved, including potential carbohydrate-specific binding proteins.
The invention comprises the following steps:
the technical problem to be solved by the invention is that the carbohydrate specific binding protein has the advantages of small molecular size, high efficiency in acquisition, flexible labeling and the like, and is an important tool for carbohydrate specific detection and analysis. There are still a large number of novel sequence, unknown functional domains present in enzymes that have not been resolved, including potential carbohydrate-specific binding proteins.
In order to solve the problems, the invention discovers that a protein sequence can be combined with pectin without being combined with other carbohydrate, has specific combination capability, and solves the problems that a structural domain with novel sequence and unknown function in pectase has specific identification and combination capability of pectin, can be separated from a catalytic domain of the pectase, can be used as independent protein, has the identification and combination functions of pectin under the condition of not degrading pectin, can be applied to pectin detection under various scenes and in-situ analysis of pectin, and has good application potential of the specific combined protein.
In order to achieve the aim, the invention is realized by the following technical scheme that the binding protein which specifically binds pectin is named as PecBP_Mp, and the amino acid sequence of the binding protein is shown as SEQ ID NO. 1.
SEQ ID NO.1：
LPPDSVYNISDMSTGTISQDTQIGDFSIIAATDGNTAIAVDGNKKTSTTTGIKYTKRLKLNGTGNQTNRAIKFTASEPATFMIEAASANSSAVRTGVLVNSAGETVASGEFASGLTYKKMTVPEAGDYWFYSTDSGINVYYLKLTYEVQPPDPVYDFEDLNGVVLEPM
The invention discovers the structural domain at the N end of pectase, which is not annotated with known functions by bioinformatics software, and is a structural domain with novel sequence and unknown functions in pectase. The inventor verifies that the gene has the function of specifically binding pectin through gene synthesis and clone expression, is a pectin specific binding protein, reveals a new gene function and represents a new protein family. The binding protein can specifically identify and directly bind to polysaccharide, and in-situ analysis of polysaccharide can be realized by fusing fluorescent probes.
The pectin-specific binding protein PecBP_Mp of the invention can specifically bind apple-derived pectin (HG), citrus peel-derived pectin (HG) and potato-derived pectin (RG-I), has no binding ability to other polysaccharides existing in cellulose, xylan, beta-glucan and other plants, and to polysaccharides containing uronic acid structures such as algin and glycosaminoglycan (shown in figure 1), and has high specificity. The PecBP_Mp is simple and convenient to prepare, low in cost, capable of being obtained in a large scale and easy to be used for specific detection of pectin.
The nucleotide sequence corresponding to the encoded pectin-specific binding protein PecBP_Mp is shown as SEQ ID NO.2, and all genes of SEQ ID NO.1 can be translated.
SEQ ID NO.2：
CTTCCGCCCGACTCTGTTTACAACATTTCTGACATGTCAACGGGCACTATCTCTCAAGATACGCAAATCGGTGATTTTTCTATTATTGCCGCTACAGACGGAAATACTGCTATTGCTGTTGACGGAAATAAAAAAACATCAACAACTACCGGAATCAAATATACAAAAAGATTAAAACTAAACGGAACCGGAAATCAAACCAACAGAGCTATAAAATTTACAGCTTCTGAGCCTGCAACATTTATGATAGAGGCAGCGAGCGCTAACTCTTCAGCTGTCAGAACAGGCGTTTTGGTAAACTCAGCCGGAGAAACAGTCGCCTCAGGCGAATTTGCGTCGGGTCTTACCTATAAAAAAATGACTGTACCCGAAGCCGGAGATTACTGGTTTTATTCTACAGACAGCGGTATAAATGTATACTATTTAAAACTTACTTATGAAGTTCAGCCGCCCGACCCGGTCTATGATTTTGAAGACCTGAACGGCGTTGTGCTTGAACCTATG
The invention provides a binding protein PecBP_Mp of specific binding pectin, which is prepared by a cloning expression method based on a definite gene sequence of the PecBP_Mp. The expression host is any one of, but not limited to, E.coli, pichia pastoris, bacillus subtilis, and the like. Comprises the following steps of:
(1) Designing primers according to the target gene, the expression vector and the enzyme cutting site, and obtaining a target gene fragment PecBP_Mp through Polymerase Chain Reaction (PCR) under the conditions of 95 ℃ for 3min,95 ℃ for 30s,52 ℃ for 30s and 72 ℃ for 60s, and circulating for 25 times.
(2) The target gene and the vector are cut by using restriction enzyme double enzyme, and are connected to form a recombinant plasmid, and then the recombinant plasmid is transferred into competent cells to form a recombinant strain. Wherein the selection of restriction enzymes and competent cells is dependent on the chosen expression vector and expression host, respectively.
(3) Strain culture, protein expression and acquisition, the conditions used in this step are selected according to the different expression hosts.
The invention also provides a method for detecting pectin based on the binding protein PecBP_Mp of the specific binding pectin. Based on the binding proteins that specifically bind pectin, the binding microarray technology enables detection of pectin.
For polysaccharide, the main function of carbohydrate active enzyme is to degrade polysaccharide, reduce the molecular weight of polysaccharide, produce active oligosaccharide, in the detection process, whether the characteristic oligosaccharide is produced can be judged by adding enzyme, but the flow is complex, the in-situ analysis can not be realized by fusing fluorescent probes, and the method has great limitation. The binding protein can be directly and specifically bound with polysaccharide, so that the polysaccharide does not need to be degraded, the molecular weight of the polysaccharide is not influenced, and in-situ analysis can be realized. In particular, the immunogenicity of saccharide substances is weak, the difficulty of preparing antibodies by animal immunization is high, and the carbohydrate specific binding protein has the advantages of small molecular size, high efficiency in acquisition, flexible labeling and the like, and is an important tool for carbohydrate specific detection and analysis. With intensive studies of carbohydrate enzymes, domains of novel sequence and unknown function have been discovered, which also include potential carbohydrate-specific binding proteins.
The first detection method comprises the following and equivalent steps:
(1) Taking 1 mu L of a sample to be tested onto a reaction medium (nitrocellulose membrane, hereinafter the reaction medium is referred to as nitrocellulose membrane), and drying at room temperature to enable the sample to be tested to be fully combined with the reaction medium.
(2) The reaction medium is blocked for 30-90min by a buffer solution (phosphate buffer solution) containing a blocking agent (skimmed milk powder) to avoid non-specific binding interference with experimental results.
(3) A quantity of pectin-specific binding protein PecBP_Mp was evenly added dropwise to the reaction medium (5 cm) 2 About 2mL binding protein for the reaction medium), for 60-120min, rinsing with water, and allowing pecbp_mp to bind to pectin.
(4) Diluting (volume ratio of 1:50000-1:5000) the anti-histidine monoclonal antibody connected with horseradish peroxidase by using the buffer solution in the step (2), and taking a certain amount of diluted antibody (5 cm) 2 About 2mL of diluted antibody) was added dropwise to the reaction medium, incubated for 60-120min, and rinsed with water. A certain amount of Electrochemiluminescence (ECL) reagent (5 cm) was added dropwise 2 About 2mL of reagent is needed for the reaction medium) to be developed for 1-2min. If pecbp_mp can bind to a polysaccharide, and because pecbp_mp contains a histidine tag, an anti-histidine monoclonal antibody linked with horseradish peroxidase can bind to pecbp_mp and adsorb to the reaction medium, and then under the catalysis of horseradish peroxidase, luminol and hydrogen peroxide in ECL reagent react to generate a fluorescent signal. In contrast, since the reaction medium was previously blocked with skimmed milk powder, if pecbp_mp had no binding ability to polysaccharide, the anti-histidine monoclonal antibody with horseradish peroxidase attached would not bind to the reaction medium and would not generate a fluorescent signal.
(5) And (5) detecting a signal. If the appearance of a fluorescent signal is observed in the imager, this is indicative of the presence of pectin in the sample; if there is no fluorescent signal, pectin is not contained.
The second detection method comprises the following steps: the pectin specific binding protein PecBP_Mp expressed by fluorescent labeling or fusion with fluorescent protein can be used in the step (3), and then the pectin can be directly detected in an imager without the step (4), so that detection and semi-quantitative analysis of pectin in a sample can be realized.
Wherein, the pectin-specific binding protein PecBP_Mp and green fluorescent protein GFP are fused (GFP-PecBP_Mp) for cloning and expression in escherichia coli and obtaining:
BamHI and NheI double enzyme cutting target gene and pRSET_EmGFP large intestine green fluorescent expression vector are connected to form recombinant plasmid. The recombinant plasmid is transferred into BL21 (DE 3) competent cells to form recombinant strain by heat shock treatment at 42 ℃. And (3) utilizing isopropyl-beta-D-thiogalactoside to induce expression, centrifugally collecting thalli, adding a certain amount of 20mM PBS buffer solution for resuspension, carrying out ice bath ultrasonic crushing, and centrifugally taking supernatant, thereby obtaining the pectin specific binding protein GFP-PecBP_Mp expressed by fluorescent protein fusion.
The method for semi-quantitatively analyzing pectin comprises the following steps: weighing pectin with different quality in the step (1), dissolving in buffer solution (phosphate buffer solution), respectively taking 1 mu L of pectin standard solution with concentration gradient on a reaction medium, drying at room temperature, and executing the steps (2) - (5); after signal detection, the fluorescence signal with known pectin concentration is converted into a gray value by using image processing software, the highest gray value (namely the highest pectin concentration) is set to be 1, and other values are standardized correspondingly. And establishing a standard curve by taking the gray value as an ordinate and the pectin concentration value as an abscissa, and carrying the gray value of the sample to be tested into the standard curve to realize semi-quantitative analysis of pectin in the sample.
Further, the pH of the buffer solution is 5 to 10. That is, the specific proteins of the present invention are suitable for use over a wide range of pH values.
The invention has the beneficial effects that:
(1) The present invention provides a novel pectin-specific binding protein pecbp_mp, which has binding capacity for pectin from a variety of different sources;
(2) The PecBP_Mp has low preparation cost and can be obtained in large scale through fermentation;
(3) The PecBP_Mp sequence in the invention is clear, flexible marking of the PecBP_Mp can be realized, and the application under different scenes can be satisfied conveniently;
(4) The invention also provides a pectin detection method based on the PecBP_Mp, which has the advantages of simple operation, strong specificity, low cost, high sensitivity and the like.
Drawings
Fig. 1: verifying the binding capacity of pecbp_mp to different polysaccharides;
fig. 2: the results of the test of example 4;
fig. 3: standard curve of example 5.
The specific embodiment is as follows:
for the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: heterologous expression of pectin-specific binding protein PecBP_Mp in E.coli
The recombinant plasmid is formed by connecting NcoI and XhoI double enzyme digestion pectin specific binding protein genes with a pET28a (+) expression vector. The recombinant plasmid is transferred into BL21 (DE 3) competent cells to form recombinant strain by heat shock treatment at 42 ℃. Positive clones were selected and induced to express by isopropyl thiogalactoside in LB medium containing kanamycin at 17 ℃ for 12h. And centrifugally collecting thalli, washing the resuspended thalli by using a buffer solution, ultrasonically crushing by using an ice bath, and centrifugally taking the supernatant.
Example 2: heterologous expression of pectin-specific binding protein PecBP_Mp in Bacillus subtilis
The target gene is cut by XbaI and SmaI and the pHT43 plasmid are connected to form a recombinant plasmid, the recombinant plasmid is transformed into bacillus subtilis competent cells, and the recombinant plasmid is cultured for 90min at 37 ℃ and then is coated on LB plates with chloramphenicol resistance. And (3) selecting positive clones, inoculating the positive clones into an LB culture medium at 37 ℃, culturing for 12 hours, inoculating the positive clones into a basic fermentation culture medium at 37 ℃ according to the inoculum size of 3%, and performing induced expression by using isopropyl thiogalactoside. The cells were collected by centrifugation, suspended in a predetermined amount of 20mM PBS buffer, and then sonicated in an ice-water bath, and the supernatant was collected by centrifugation.
Example 3: heterologous expression of pectin-specific binding protein PecBP_MP in Pichia pastoris
The EcoRI and NotI double enzyme cutting target genes are utilized to be connected with pPIC9k plasmid to form recombinant plasmid, and after Sac I enzyme cutting, the recombinant plasmid is transformed into pichia pastoris GS115 competent cells to form recombinant cells. Single colonies were extracted for PCR validation. The single clone after the correct sequencing and antibiotic screening was cultured in YPD liquid medium at 30℃and 220rpm for 12 hours. Then inoculating to BMGY culture medium with pH of 6.0, culturing at 30deg.C and 220rpm until OD600 is 5.0, centrifuging, adding BMMY culture medium with pH of 6.0, culturing, adding 0.5% methanol at 29 deg.C and 220rpm, and inducing expression for 72 hr. The supernatant was collected by centrifugation.
Example 4: detecting whether pectin is contained in beverage containing pectin component in label
(1) Respectively taking 1 mu L of beverage to be measured and ultrapure water onto a nitrocellulose membrane, and drying for more than 30 minutes at room temperature for three times in parallel;
(2) The nitrocellulose membrane was blocked with phosphate buffer (140 mM sodium chloride, 2.7mM potassium chloride, 10mM disodium hydrogen phosphate, 1.7mM potassium dihydrogen phosphate, pH 7.5) containing 5% nonfat milk powder for 1 hour, and rinsed with ultrapure water as appropriate;
(3) Taking 2mL of pectin specific binding protein PecBP_Mp, uniformly dripping the PecBP_Mp onto a nitrocellulose membrane, incubating for 2h, and flushing with ultrapure water;
(4) Diluting (1:5000) the anti-histidine monoclonal antibody connected with horseradish peroxidase by using the phosphate buffer solution containing 5% of skimmed milk powder in the step (2), taking 2mL of the anti-histidine monoclonal antibody, uniformly dripping the anti-histidine monoclonal antibody onto a nitrocellulose membrane, incubating for 2h, and flushing with ultrapure water;
(5) Uniformly dripping 2mL of ECL reagent onto the nitrocellulose membrane, and developing for 2min;
(6) And detecting a chemiluminescent instrument signal.
As can be seen from the results of the experiment in FIG. 2, the beverage contains pectin.
Example 5: pectin in semi-quantitative freshly squeezed apple juice
(1) Respectively preparing apple pectin standard solutions (purchased reagent-grade apple pectin) with different concentrations, namely 20mg/mL, 16mg/mL, 12mg/mL, 8mg/mL, 4mg/mL and 2mg/mL, respectively sucking 1 mu L of the standard solutions with different concentrations and freshly squeezed apple juice to be detected onto a nitrocellulose membrane for three times in parallel, and drying at room temperature for more than 30 min;
(2) The nitrocellulose membrane was blocked with phosphate buffer (140 mM sodium chloride, 2.7mM potassium chloride, 10mM disodium hydrogen phosphate, 1.7mM potassium dihydrogen phosphate, pH 7.5) containing 5% nonfat milk powder for 1 hour, and rinsed with ultrapure water as appropriate;
(3) Uniformly dripping 4mL of pectin specific binding protein PecBP_Mp on a nitrocellulose membrane, incubating for 2h, and flushing with ultrapure water;
(4) Diluting (1:5000) the anti-histidine monoclonal antibody connected with horseradish peroxidase by using the phosphate buffer solution of 5% of skimmed milk powder contained in the step (2), taking 4mL of the anti-histidine monoclonal antibody, uniformly dripping the anti-histidine monoclonal antibody onto a nitrocellulose membrane, incubating for 2h, and flushing with ultrapure water;
(5) Uniformly dripping 4mL of ECL reagent on the nitrocellulose membrane, and developing for 2min;
(6) Detecting signals by using a chemiluminescent instrument, converting fluorescent signals with known apple pectin concentration into gray values by using imageJ image processing software, setting the maximum concentration value (namely 20 mg/mL) as 1, correspondingly standardizing other values, substituting the gray values of a sample to be detected into a standard curve y=0.0458x+0.1355, and R 2 = 0.9803 (this standard curve is applicable at pectin concentrations of 2-20 mg/mL). Thus realizing semi-quantitative analysis of pectin in freshly squeezed apple juice.
The pectin concentration in the freshly squeezed apple juice is about 7.90mg/mL.
Wherein the binding specificity of the binding protein has been demonstrated in fig. 1, and thus the signal detected on the nitrocellulose membrane is the specific binding signal of the protein to pectin. The purpose of example 4 is to detect the authenticity of a label component, and can be applied to pectin component detection in various other scenes such as functional foods, cosmetics and the like. Also, in example 5, it was demonstrated that the method does not generate binding signals to cellulose and the like that may be present in freshly squeezed juice, due to the demonstrated binding specificity.
The above examples are only intended to illustrate the technical scheme of the present invention, but not to limit the invention, and other reagents and raw materials of unspecified origin are commercially available. Although the invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that modifications and equivalents may be made to the present invention without departing from the spirit and scope of the invention.

Claims (10)

1. The amino acid sequence of the binding protein specifically binding to pectin is shown as SEQ ID NO. 1.
2. The nucleotide sequence corresponding to the coded binding protein of claim 1 is shown as SEQ ID NO.2, and all genes of SEQ ID NO.1 can be translated.
3. The method for preparing the binding protein according to claim 1, wherein: based on the gene sequence of the specific binding protein, the binding protein of the specific binding pectin is prepared by a cloning expression method.
4. Use of the binding protein of claim 1 for detection and semi-quantitative analysis of pectin.
5. A method for detecting and semi-quantitatively analyzing pectin based on the binding protein of claim 1, characterized in that: detection of pectin is achieved by binding microarray technology based on binding proteins that specifically bind pectin.
6. The method of detection according to claim 5, wherein the method of detection comprises the steps of:
(1) Taking 1 mu L of a sample to be tested to a reaction medium, and drying the sample at room temperature, wherein the sample to be tested is fully combined with the reaction medium;
(2) Sealing the reaction medium with a buffer solution containing a sealing agent for 30-90min;
(3) Uniformly dripping pectin specific binding protein PecBP_Mp onto a reaction medium, incubating, and washing with water to enable the PecBP_Mp to be bound with pectin;
(4) Diluting the anti-histidine monoclonal antibody connected with horseradish peroxidase by using the buffer solution in the step (2), taking a certain amount of diluted antibody, uniformly dripping the diluted antibody onto a reaction medium, and washing with water after incubation; uniformly dripping a certain amount of electrochemiluminescence reagent onto the reaction medium, and developing for 1-2min;
(5) And (3) signal detection: if the appearance of a fluorescent signal is observed in the imager, this is indicative of the presence of pectin in the sample; if there is no fluorescent signal, pectin is not contained.
7. The method of detection according to claim 5, wherein the method of detection comprises the steps of:
(1) Taking 1 mu L of a sample to be tested to a reaction medium, and drying the sample at room temperature, wherein the sample to be tested is fully combined with the reaction medium;
(2) Sealing the reaction medium with a buffer solution containing a sealing agent for 30-90min;
(3) Uniformly dripping pectin specific binding protein PecBP_Mp which is marked by fluorescence or fused and expressed with the fluorescent protein onto a reaction medium, and washing with water after incubation to combine the fused and expressed PecBP_Mp with pectin;
(4) And (3) signal detection: if the appearance of a fluorescent signal is observed in the imager, this is indicative of the presence of pectin in the sample; if there is no fluorescent signal, pectin is not contained.
8. The method of detection according to claim 5, wherein the semi-quantitative analysis method comprises the steps of:
(1) Weighing pectin with different masses, dissolving in buffer solution, respectively weighing 1 μl pectin standard solution with concentration gradient on reaction medium, and drying at room temperature; taking 1 mu L of sample to be tested on the reaction medium, and drying at room temperature;
(2) Sealing the reaction medium with buffer solution containing sealing agent for 30-90min;
(3) Uniformly dripping pectin specific binding protein PecBP_Mp onto each reaction medium, incubating, and washing with water to enable the PecBP_Mp to be bound with pectin;
(4) Diluting the anti-histidine monoclonal antibody connected with horseradish peroxidase by using the buffer solution in the step (2), taking a certain amount of diluted antibody, uniformly dripping the diluted antibody onto each reaction medium, and washing with water after incubation; uniformly dripping a certain amount of electrochemiluminescence reagent onto the reaction medium, and developing for 1-2min;
(5) And (3) signal detection: converting the fluorescence signal with known pectin concentration into a gray value by using image processing software, setting the highest gray value to be 1, and correspondingly standardizing other values; and establishing a standard curve by taking the gray value as an ordinate and the pectin concentration value as an abscissa, and carrying the gray value of the sample to be tested into the standard curve to realize semi-quantitative analysis of pectin in the sample.
9. The method of any one of claims 6-8, wherein: the pH value of the buffer solution is 5-10.
10. The method of any one of claims 6 or 8, wherein: in the step (4), the buffer solution is used according to the volume ratio of 1:50000-1:5000 ratio.
CN202311572990.1A 2023-11-23 2023-11-23 Binding protein capable of specifically binding pectin and preparation and application thereof Pending CN117603953A (en)

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