Affinity purification method based on non-natural amino acid protein

Abstract

The invention discloses a protein affinity purification method based on non-natural amino acid, which is applied to the field of protein affinity purification, and comprises the following steps: selecting proper non-natural amino acid and sites, optimizing codons, constructing a gene vector and transforming cells, optimizing cell culture conditions, and formulating a proper inducible expression strategy. The expression process is monitored in real time, the specific affinity ligand is prepared, the combination, washing and elution conditions are optimized, and the target protein is captured. The non-natural amino acid is introduced into the target protein at a fixed point through genetic engineering, for example, the non-natural amino acid with an azido group is introduced, integration of the specific non-natural amino acid in cells has high specificity, and compared with a traditional natural amino acid tag, the possibility of non-specific labeling is greatly reduced, and the application prospect is wide. Therefore, co-purification of impurities in the purification process is reduced.

Description

Technical Field

[0001] The present invention relates to the field of protein affinity purification, and in particular, to a method for protein affinity purification based on non-natural amino acids. Background Art

[0002] Protein is a key molecule in life activities, and its purification is one of the core tasks in the fields of biochemistry, molecular biology and biotechnology.

[0003] Traditional protein purification methods have the problems of low specificity, low purification efficiency, easy loss of protein activity, difficulty in removing similar impurities, and lack of real-time monitoring and adjustment mechanisms.

[0004] There is no effective solution to the problems in the related technology. Therefore, this case proposes a method for protein affinity purification based on non-natural amino acids. Summary of the invention

[0005] In order to overcome the above problems, the present invention aims to propose a method for protein affinity purification based on non-natural amino acids, with the aim of solving the problems of traditional protein purification methods such as low specificity, low purification efficiency, easy loss of protein activity, difficulty in removing similar impurities, and lack of real-time monitoring and adjustment mechanisms.

[0006] To this end, the specific technical solution adopted by the present invention is as follows:

[0007] According to one aspect of the present invention, a method for protein affinity purification based on non-natural amino acids is provided, the purification method comprising the following steps:

[0008] S1. Select appropriate unnatural amino acids and sites, optimize codons, construct gene vectors and transform cells;

[0009] S2. Optimize cell culture conditions, develop appropriate induction expression strategies, and monitor the expression process in real time;

[0010] S3, prepare specific affinity ligands, optimize binding, washing and elution conditions, and capture target proteins;

[0011] S4, using affinity purification method to gradually remove various impurities and finely separate the target protein;

[0012] S5, real-time collection and analysis of data during the purification process, and adjustment of purification conditions based on the results;

[0013] S6. Collect the purified protein, concentrate and store it, and identify the purity, structure and activity of the protein.

[0014] Optionally, the specific process of selecting suitable non-natural amino acids and sites, optimizing codons, constructing gene vectors and transforming cells is as follows:

[0015] S11: Evaluate the exposure degree of different sites by calculating the solvent accessible surface area of ​​the protein, use energy minimization algorithm and molecular dynamics simulation to evaluate the structural stability after the incorporation of unnatural amino acids at different sites, select sites with suitable chemical environment and spatial structure, and ensure that the incorporation of unnatural amino acids will not significantly affect the folding and activity of the protein;

[0016] Formula: SASA = ∑(0.5*π*r 2 ), where r is the atomic radius;

[0017] S12: According to the codon usage preference of the host cell, the codons for introducing non-natural amino acids are optimized to increase the translation efficiency. For example, in Escherichia coli, the use of high-frequency codons can increase the translation speed. The target protein gene fragment containing the optimized codons is synthesized by chemical synthesis or gene splicing. During the gene synthesis process, the gene is detected by agarose gel electrophoresis or sequencing to ensure the accuracy and integrity of the gene.

[0018] Formula: Translation efficiency = codon usage frequency / total codon occurrence frequency;

[0019] S13: Insert the synthesized gene fragment into any one or more expression vectors such as plasmids and viral vectors to construct a recombinant expression vector. The selected vector should have promoter, terminator and selection marker elements suitable for the host cell. Use electroporation, chemical transformation or liposome transfection to introduce the recombinant vector into the host cell, and select the successfully transformed cell strain through the selection marker (such as antibiotic resistance).

[0020] Optionally, the specific process of optimizing cell culture conditions, formulating an appropriate induction expression strategy, and monitoring the expression process in real time is as follows:

[0021] S21: Optimize the composition of the cell culture medium, including carbon source, nitrogen source, vitamins and minerals, to meet the needs of cell growth and protein expression, add precursor substances and auxiliary factors, promote the uptake and incorporation of non-natural amino acids, control the culture environment parameters, temperature 35-37°C, pH 7.2-7.4, air saturation 20%-80%, stirring speed 100-200rpm, use online monitoring equipment to monitor these parameters in real time, and adjust them through a feedback control system, calculate the cell growth rate according to cell growth rate = (lnX2-lnX1) / (t2-t1), where X1 and X2 are the cell concentrations at the start and end, respectively, and t1 and t2 are the corresponding times;

[0022] S22: During the induction expression process, samples are taken regularly to analyze the protein quality by SDS-PAGE, Western blotting, or mass spectrometry to monitor the expression level, molecular weight, and modification status of the target protein. The inducer concentration and induction time are optimized based on the analysis results to ensure efficient expression and correct folding of the target protein.

[0023] Optionally, the specific process of preparing a specific affinity ligand, optimizing binding, washing and elution conditions, and capturing the target protein is as follows:

[0024] S31: Based on the chemical structure and reaction characteristics of the selected non-natural amino acid, an affinity ligand with high specificity and affinity is synthesized, and the affinity calculation formula is: affinity constant (Kd) = [L] * [R] / [LR], where [L] is the free ligand concentration, [R] is the free receptor concentration, and [LR] is the ligand-receptor complex concentration, such as a non-natural amino acid with an azide group, synthesizing an affinity ligand with a cycloalkyne group, preparing the affinity ligand by chemical synthesis or biosynthesis, and using liquid chromatography technology to detect the purity and identify the structure of the ligand;

[0025] S32: Detect the pH value, ionic strength, temperature and reaction time of the buffer between the affinity ligand and the non-natural amino acid. During the affinity binding process, add appropriate surfactants or blocking agents to inhibit nonspecific adsorption and improve the selectivity of affinity purification.

[0026] S33: Select buffers with different ionic strengths and pH values ​​for washing to remove non-specifically adsorbed impurities and weakly bound molecules, and use specific cleavage reagents to elute the target protein.

[0027] Optionally, the affinity purification method is used to gradually remove various impurities and finely separate the target protein. The specific process is as follows:

[0028] S41: Use specific affinity ligands for non-natural amino acids for preliminary purification to remove most non-target proteins and impurities, and perform quality testing on the samples after preliminary purification. The testing items include but are not limited to measuring protein concentration, purity and activity to obtain the purification effect;

[0029] S42: Based on the characteristics of the sample and the type of impurities after purification in S41, further fine purification is performed through ion exchange chromatography, and the quality inspection steps are repeated. If the inspection results do not meet the standards, the purification steps are repeated.

[0030] Optionally, the real-time data collection and analysis during the purification process and the purification conditions adjustment according to the results are specifically performed as follows;

[0031] S51: Install any one or more of online fluorescence detection devices, high performance liquid chromatography and mass spectrometry to monitor the target protein and impurities in real time, establish a data acquisition and transmission system, and transmit the real-time detection data to the central control computer;

[0032] S52: Preprocess the raw test data using data smoothing, baseline correction and peak identification algorithms to remove noise and interference signals, and analyze the processed data using partial least squares regression statistical analysis and decision tree machine learning algorithms to extract key information, including but not limited to protein concentration change trends and impurity accumulation patterns;

[0033] S53: Based on the data analysis results, formulate feedback control strategies, including online monitoring to detect decreased protein purity, increase the number or intensity of washing steps, and adjust the flow rate or extend the binding time if the protein concentration is too low.

[0034] Optionally, the specific process of collecting the purified protein, concentrating and storing it, and identifying the purity, structure and activity of the protein is as follows:

[0035] S61: collecting the purified protein solution, concentrating and desalting the solution by ultrafiltration, dialysis or freeze drying, and storing the solution at low temperature;

[0036] S62: Use high performance liquid chromatography (HPLC) to identify the purity of the collected proteins, determine the molecular weight, amino acid composition and modification of the proteins, use circular dichroism (CD) to detect the protein structure, and design activity assays for the target proteins, such as enzyme activity assays, binding activity assays or cell biology experiments, to verify whether the purified proteins maintain the expected biological activity and function.

[0037] Compared with the prior art, this application has the following beneficial effects:

[0038] 1. Introducing non-natural amino acids into target proteins through genetic engineering, such as introducing non-natural amino acids with azide groups. This specific non-natural amino acid is highly specific in its integration into cells. Compared with traditional natural amino acid tags, it greatly reduces the possibility of non-specific labeling, thereby reducing the co-purification of impurities during the purification process.

[0039] 2. Design customized affinity ligands for non-natural amino acids, such as small molecule ligands with cycloalkyne groups, which undergo efficient and specific click chemistry reactions with non-natural amino acids (such as azide groups) to form stable and specific binding. Compared with traditional affinity ligands, this customized design can more accurately identify and bind to target proteins, significantly improve the selectivity and efficiency of purification, and reduce cross-reactions with other proteins with similar structures.

[0040] 3. A multi-step affinity purification strategy is adopted. First, affinity ligands for non-natural amino acids are used for preliminary purification to remove most non-target proteins and impurities. Then, according to the physicochemical properties of the target protein (such as charge, molecular weight, hydrophobicity, etc.), appropriate traditional purification methods (such as ion exchange chromatography, gel filtration chromatography, hydrophobic interaction chromatography, etc.) are selected for further fine purification. Each step of purification removes specific types of impurities, thereby gradually improving the purity of the protein.

[0041] 4. Introduce online monitoring technologies, such as real-time fluorescence detection, high-performance liquid chromatography and mass spectrometry, collect samples in real time and analyze them during the purification process, which can monitor the concentration, purity and impurity content of the target protein in real time. Based on these real-time data, an automated feedback control system is used to timely adjust the parameters of the purification process (such as flow rate, buffer composition, temperature, etc.). This real-time monitoring and adjustment mechanism can optimize the purification effect and ensure that each batch of purification can achieve high quality and consistency. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The above characteristics, features and advantages of the present invention and their implementation methods and methods will become more clearly understood in conjunction with the following description of the embodiments, which are described in detail in conjunction with the accompanying drawings. Herein, a schematic diagram is shown:

[0043] Figure 1 The present invention is a flowchart of a method for protein affinity purification based on non-natural amino acids. DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0045] According to an embodiment of the present invention, a method for protein affinity purification based on non-natural amino acids is provided.

[0046] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. Figure 1 As shown, according to one embodiment of the present invention, a method for protein affinity purification based on non-natural amino acids is provided, and the purification method comprises the following steps:

[0047] S1. Select appropriate unnatural amino acids and sites, optimize codons, construct gene vectors and transform cells. The specific process is as follows:

[0048] S11: Evaluate the exposure degree of different sites by calculating the solvent accessible surface area of ​​the protein, use energy minimization algorithm and molecular dynamics simulation to evaluate the structural stability after the incorporation of unnatural amino acids at different sites, select sites with suitable chemical environment and spatial structure, and ensure that the incorporation of unnatural amino acids will not significantly affect the folding and activity of the protein;

[0049] Formula: SASA = ∑(0.5*π*r 2 ), where r is the atomic radius;

[0050] S12: According to the codon usage preference of the host cell, the codons for introducing non-natural amino acids are optimized to increase the translation efficiency. For example, in Escherichia coli, the use of high-frequency codons can increase the translation speed. The target protein gene fragment containing the optimized codons is synthesized by chemical synthesis or gene splicing. During the gene synthesis process, the gene is detected by agarose gel electrophoresis or sequencing to ensure the accuracy and integrity of the gene.

[0051] Formula: Translation efficiency = codon usage frequency / total codon occurrence frequency;

[0052] S13: inserting the synthesized gene fragment into any one or more expression vectors such as plasmids and viral vectors to construct a recombinant expression vector. The selected vector should have a promoter, terminator and selection marker element suitable for the host cell. The recombinant vector is introduced into the host cell by any one of electroporation, chemical transformation or liposome transfection, and the successfully transformed cell strain is selected by the selection marker (such as antibiotic resistance);

[0053] S2. Optimize cell culture conditions, develop appropriate induction expression strategies, and monitor the expression process in real time. The specific process is as follows:

[0054] S21: Optimize the composition of the cell culture medium, including carbon source, nitrogen source, vitamins and minerals, to meet the needs of cell growth and protein expression, add precursor substances and auxiliary factors, promote the uptake and incorporation of non-natural amino acids, control the culture environment parameters, temperature 35-37°C, pH 7.2-7.4, air saturation 20%-80%, stirring speed 100-200rpm, use online monitoring equipment to monitor these parameters in real time, and adjust them through a feedback control system, calculate the cell growth rate according to cell growth rate = (lnX2-lnX1) / (t2-t1), where X1 and X2 are the cell concentrations at the start and end, respectively, and t1 and t2 are the corresponding times;

[0055] S22: During the induction expression process, samples are taken regularly to analyze the protein quality by SDS-PAGE, Western blotting, or mass spectrometry to monitor the expression level, molecular weight, and modification status of the target protein. The inducer concentration and induction time are optimized based on the analysis results to ensure efficient expression and correct folding of the target protein.

[0056] S3. Prepare specific affinity ligands, optimize binding, washing and elution conditions, and capture target proteins. The specific process is as follows:

[0057] S31: Based on the chemical structure and reaction characteristics of the selected non-natural amino acid, an affinity ligand with high specificity and affinity is synthesized, and the affinity calculation formula is: affinity constant (Kd) = [L] * [R] / [LR], where [L] is the free ligand concentration, [R] is the free receptor concentration, and [LR] is the ligand-receptor complex concentration, such as a non-natural amino acid with an azide group, synthesizing an affinity ligand with a cycloalkyne group, preparing the affinity ligand by chemical synthesis or biosynthesis, and using liquid chromatography technology to detect the purity and identify the structure of the ligand;

[0058] S32: Detect the pH value, ionic strength, temperature and reaction time of the buffer between the affinity ligand and the non-natural amino acid. During the affinity binding process, add appropriate surfactants or blocking agents to inhibit nonspecific adsorption and improve the selectivity of affinity purification.

[0059] S33: Select buffers with different ionic strengths and pH values ​​for washing to remove non-specifically adsorbed impurities and weakly bound molecules, and use specific cleavage reagents to elute the target protein;

[0060] S4. Use affinity purification method to gradually remove various impurities and finely separate the target protein. The specific process is as follows:

[0061] S41: Use specific affinity ligands for non-natural amino acids for preliminary purification to remove most non-target proteins and impurities, and perform quality testing on the samples after preliminary purification. The testing items include but are not limited to measuring protein concentration, purity and activity to obtain the purification effect;

[0062] S42: Based on the characteristics of the sample and the type of impurities after purification in S41, further fine purification is performed by ion exchange chromatography, and the quality inspection step is repeated. If the inspection result does not meet the standard, the purification step is repeated;

[0063] S5. Real-time data collection and analysis during the purification process, and adjustment of purification conditions according to the results. The specific process is as follows;

[0064] S51: Install any one or more of online fluorescence detection devices, high performance liquid chromatography and mass spectrometry to monitor the target protein and impurities in real time, establish a data acquisition and transmission system, and transmit the real-time detection data to the central control computer;

[0065] S52: Preprocess the raw test data using data smoothing, baseline correction and peak identification algorithms to remove noise and interference signals, and analyze the processed data using partial least squares regression statistical analysis and decision tree machine learning algorithms to extract key information, including but not limited to protein concentration change trends and impurity accumulation patterns;

[0066] S53: Based on the data analysis results, develop feedback control strategies, including increasing the number or intensity of washing steps if protein purity decreases during online monitoring, adjusting the flow rate or extending the binding time if the protein concentration is too low;

[0067] S6. Collect the purified protein, concentrate and store it, and identify the purity, structure and activity of the protein. The specific process is as follows:

[0068] S61: collecting the purified protein solution, concentrating and desalting the solution by ultrafiltration, dialysis or freeze drying, and storing the solution at low temperature;

[0069] S62: The collected proteins were purified by high performance liquid chromatography (HPLC), and their molecular weight, amino acid composition, and modification were determined. Circular dichroism (CD) was used to detect the protein structure.

[0070] S63: Design activity assays for the target protein, such as enzyme activity assays, binding activity assays, or cell biology experiments, to verify whether the purified protein maintains the expected biological activity and function.

[0071] It should be noted that the possible unsatisfactory performance, causes and solutions of the detailed method of protein affinity purification based on non-natural amino acids are shown in Table 1:

[0072] Table 1

[0073]

[0074]

[0075]

[0076] Although the present invention has been disclosed as above with preferred embodiments, the embodiments are merely examples for the convenience of description and are not intended to limit the present invention. Those skilled in the art may make several changes and modifications without departing from the spirit and scope of the present invention. The scope of protection claimed by the present invention shall be based on the claims.

Claims

1. A method for protein affinity purification based on non-natural amino acids, characterized in that: The purification method comprises the following steps: S1. Select appropriate unnatural amino acids and sites, optimize codons, construct gene vectors and transform cells; S2. Optimize cell culture conditions, develop appropriate induction expression strategies, and monitor the expression process in real time; S3, prepare specific affinity ligands, optimize binding, washing and elution conditions, and capture target proteins; S4, using affinity purification method to gradually remove various impurities and finely separate the target protein; S5, real-time collection and analysis of data during the purification process, and adjustment of purification conditions based on the results; S6. Collect the purified protein, concentrate and store it, and identify the purity, structure and activity of the protein.

2. A method for protein affinity purification based on non-natural amino acids according to claim 1, characterized in that: The specific process of selecting suitable non-natural amino acids and sites, optimizing codons, constructing gene vectors and transforming cells is as follows: S11: Evaluate the exposure degree of different sites by calculating the solvent accessible surface area of ​​the protein, use energy minimization algorithm and molecular dynamics simulation to evaluate the structural stability after incorporation of unnatural amino acids at different sites, and select sites with suitable chemical environment and spatial structure; Formula: SASA = ∑(0.5*π*r 2 ), where r is the atomic radius; S12: According to the codon usage preference of the host cell, the codons for introducing non-natural amino acids are optimized to increase the translation efficiency, and the target protein gene fragment containing the optimized codons is synthesized by chemical synthesis or gene splicing. During the gene synthesis process, detection is performed by agarose gel electrophoresis or sequencing. Formula: Translation efficiency = codon usage frequency / total codon occurrence frequency; S13: Insert the synthesized gene fragment into any one or more expression vectors such as plasmids and viral vectors to construct a recombinant expression vector, introduce the recombinant vector into the host cell using any one of electroporation, chemical transformation or liposome transfection, and screen out the successfully transformed cell lines using the screening marker.

3. A method for protein affinity purification based on non-natural amino acids according to claim 1, characterized in that: The specific process of optimizing cell culture conditions, formulating appropriate induction expression strategy, and real-time monitoring of the expression process is as follows: S21: Optimize the composition of the cell culture medium, including carbon source, nitrogen source, vitamins and minerals, to meet the needs of cell growth and protein expression, add precursor substances and auxiliary factors, promote the uptake and incorporation of non-natural amino acids, control the culture environment parameters, temperature 35-37°C, pH 7.2-7.4, air saturation 20%-80%, stirring speed 100-200rpm, and calculate the cell growth rate according to cell growth rate = (lnX2-lnX1) / (t2-t1), where X1 and X2 are the cell concentrations at the start and end, respectively, and t1 and t2 are the corresponding times; S22: During the induction expression process, samples are taken regularly to analyze the protein quality by SDS-PAGE, Western blotting, or mass spectrometry to monitor the expression level, molecular weight, and modification status of the target protein, and the inducer concentration and induction time are optimized based on the analysis results.

4. A method for protein affinity purification based on non-natural amino acids according to claim 1, characterized in that: The specific process of preparing the specific affinity ligand, optimizing the binding, washing and elution conditions, and capturing the target protein is as follows: S31: synthesizing an affinity ligand with high specificity and affinity according to the chemical structure and reaction characteristics of the selected non-natural amino acid, wherein the affinity calculation formula is: affinity constant (Kd) = [L]*[R] / [LR], wherein [L] is the free ligand concentration, [R] is the free receptor concentration, and [LR] is the ligand-receptor complex concentration, and the affinity ligand is prepared by chemical synthesis or biosynthesis; S32: Detect the pH value, ionic strength, temperature and reaction time of the buffer between the affinity ligand and the non-natural amino acid. During the affinity binding process, add appropriate surfactants or blocking agents to inhibit nonspecific adsorption. S33: Select buffers with different ionic strengths and pH values ​​for washing to remove non-specifically adsorbed impurities and weakly bound molecules, and use specific cleavage reagents to elute the target protein.

5. A method for protein affinity purification based on non-natural amino acids according to claim 1, characterized in that: The affinity purification method is used to gradually remove various impurities and finely separate the target protein. The specific process is as follows: S41: Use specific affinity ligands for non-natural amino acids for preliminary purification to remove most non-target proteins and impurities, and perform quality testing on the samples after preliminary purification. The testing items include but are not limited to measuring protein concentration, purity and activity to obtain the purification effect; S42: Based on the characteristics of the sample and the type of impurities after purification in S41, further fine purification is performed through ion exchange chromatography, and the quality inspection steps are repeated. If the inspection results do not meet the standards, the purification steps are repeated.

6. A method for protein affinity purification based on non-natural amino acids according to claim 1, characterized in that: The specific process of collecting and analyzing the data in the purification process in real time and adjusting the purification conditions according to the results is as follows; S51: Install any one or more of online fluorescence detection devices, high performance liquid chromatography and mass spectrometry to monitor the target protein and impurities in real time, establish a data acquisition and transmission system, and transmit the real-time detection data to the central control computer; S52: Preprocess the raw test data using data smoothing, baseline correction and peak identification algorithms to remove noise and interference signals, and analyze the processed data using partial least squares regression statistical analysis and decision tree machine learning algorithms to extract key information, including but not limited to protein concentration change trends and impurity accumulation patterns; S53: Based on the data analysis results, formulate feedback control strategies, including online monitoring to detect decreased protein purity, increase the number or intensity of washing steps, and adjust the flow rate or extend the binding time if the protein concentration is too low.

7. A method for protein affinity purification based on non-natural amino acids according to claim 6, characterized in that: The specific process of collecting the purified protein, concentrating and storing it, and identifying the purity, structure and activity of the protein is as follows: S61: collecting the purified protein solution, concentrating and desalting the solution by ultrafiltration, dialysis or freeze drying, and storing the solution at low temperature; S62: The collected proteins were purified by high performance liquid chromatography (HPLC) to determine their molecular weight, amino acid composition and modification, and circular dichroism (CD) was used to detect their structure.

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