WO2020181756A1 - 基于α螺旋把手提高蛋白核酸生物耦合效率的方法 - Google Patents

基于α螺旋把手提高蛋白核酸生物耦合效率的方法 Download PDF

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WO2020181756A1
WO2020181756A1 PCT/CN2019/107289 CN2019107289W WO2020181756A1 WO 2020181756 A1 WO2020181756 A1 WO 2020181756A1 CN 2019107289 W CN2019107289 W CN 2019107289W WO 2020181756 A1 WO2020181756 A1 WO 2020181756A1
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protein
tag
nucleic acid
amino acid
unnatural amino
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于仲波
李旭
马小凤
屈利花
黄威
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南开大学
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

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  • the invention belongs to the field of medicinal chemistry biology, and in particular relates to a method for improving the efficiency of protein nucleic acid biological coupling based on an alpha helix handle.
  • biological coupling is an important tool for studying the interaction of biological macromolecules. It is of great significance for a deep understanding of the functions of macromolecules in cell life activities and the dynamic mechanism of drugs in regulating the biological activities of macromolecules. It has become a drug, chemistry and biology. The cutting-edge direction of the cross field.
  • Biological coupling can occur between protein and protein, protein and nucleic acid, nucleic acid and nucleic acid, and drug and protein nucleic acid.
  • Commonly used biological coupling reactions include click chemistry, disulfide bond and maleimide reactions.
  • the groups of natural amino acids involved in protein coupling include amino, carboxyl and sulfhydryl groups.
  • Natural amino acids have many sites for modification groups, high overall reactivity, and easy to carry out biological coupling; but the disadvantage is that site selection specificity is low and site-specific modification cannot be performed.
  • Bio-orthogonal technology allows cells to use translation machinery to introduce unnatural amino acids into proteins at specific points. This breaks through the limitations of the selection of natural amino acid biological coupling groups, and greatly enriches the library of modified groups.
  • unnatural amino acids can be introduced at any position of the protein through site-directed genetic mutation, the complex nano-environment on the surface of the protein makes it difficult to predict the reactivity of the unnatural amino acid coupling group.
  • the main factors that affect the reactivity of the unnatural amino acid coupling group include the solvent exposure of the introduction site, the charge level of the amino acid itself and surroundings, and the polarity characteristics of the adjacent amino acid groups.
  • Protein coupling through unnatural amino acids requires complicated mutation site optimization and selection procedures, and faces the dilemma of low protein coupling efficiency.
  • the biological coupling of protein and nucleic acid is essential for studying the interaction between protein and nucleic acid. Therefore, it is very necessary to develop a universal handle carrying unnatural amino acid introduction sites for efficient biological coupling of proteins and nucleic acids.
  • the present invention provides a method for improving the efficiency of protein nucleic acid biological coupling based on an alpha helix handle, which is used to optimize the design of protein coupling group positions, especially the detection of biological macromolecule interactions based on coupling reactions.
  • the technical scheme adopted by the present invention is: based on the method of ⁇ -helix handle to increase the efficiency of protein nucleic acid biological coupling, the ⁇ -helix handle is connected with the end of the protein using a connecting polypeptide, and the unnatural amino acid is inserted into the specific position of the ⁇ -helix handle by using the extended genetic code. Point, through click chemistry, the efficient biological coupling of protein and nucleic acid is realized.
  • Step 1 Based on the alpha helix of the protein secondary structure, design a handle H-tag carrying unnatural amino acids and connect it to one end of the protein to be tested through a polypeptide;
  • Step 2 Mutation of a codon of the handle H-tag into an unnatural amino acid codon by site-directed genetics, construct a fusion recombinant expression plasmid containing the H-tag and the protein to be tested through a prokaryotic expression vector, and transfer it into competent cells and then screen to obtain Stable genetic clone strain;
  • Step 3 Co-transform the fusion recombinant expression plasmid containing the H-tag and the protein to be tested and the plasmid capable of expressing tRNA/aminoacyl tRNA synthetase to obtain the prokaryotic expression strain, add unnatural amino acids to the medium, and induce the expression fusion with an inducer
  • the H-tag protein to be tested
  • Step 4 lyse the cells of the prokaryotic expression strain after the induction of expression, and purify the soluble protein of H-tag-the protein to be tested with the aid of the affinity tag of the recombinant protein;
  • Step 5 Use click chemistry to efficiently connect the coupling group on the H-tag-test protein and the nucleic acid substrate, and use gel electrophoresis to detect the reaction efficiency.
  • the unnatural amino acid is an unnatural amino acid coupled to an azide group, specifically azidophenylalanine.
  • click chemistry is a tension-driven azide-alkynyl cyclization reaction.
  • the length of the H-tag in step 1 is greater than 5 amino acids and is designed at the amino or carboxyl end of the protein to be tested;
  • the length of the connecting polypeptide is greater than 8 amino acids
  • the preferred position of the unnatural amino acid in the H-tag sequence satisfies the conditions of neutrality in electricity, polar amino acid environment and relative solvent accessibility of the amino acid value.
  • the prokaryotic expression vector in step 2 is a pET series vector
  • the unnatural amino acid codon is an amber codon
  • the fusion recombinant expression plasmid containing the H-tag and the protein to be tested can be constructed by a cloning method that depends on ligation or a seamless cloning method.
  • the medium in step 3 contains 5% glycerol and 1 mM unnatural amino acid
  • the inducer uses 1 mM isopropyl- ⁇ -D-thiogalactoside (IPTG);
  • the induced expression time is preferably 12-20h, preferably 16h;
  • the orthogonal pair encoding genes in the plasmid capable of expressing tRNA/aminoacyl tRNA synthetase can be single copy or multiple copies.
  • the cells are lysed under the action of a protease inhibitor
  • the protease inhibitor is preferably phenylmethylsulfonyl fluoride at a concentration of 0.1-1 mM
  • the affinity tag is hexahistidine or glutathione sulfhydryl transferase
  • an ultrasonic disruptor is used to lyse cells, the power is set to 200W, the cycle is on for 4 seconds and off for 6 seconds, and the total time is set to 25-40 min;
  • the protein is purified using chromatography technology, preferably affinity chromatography, specifically metal chelation affinity chromatography or combined use of ion exchange chromatography.
  • the coupling group on the nucleic acid substrate in step 5 is a polyethylene glycol diphenyl cyclooctyne group
  • the molar ratio of the fusion protein to the nucleic acid substrate is 1:2.5-5, preferably 1:5;
  • the reaction temperature is 12°C, and the reaction time is 24h;
  • the gel electrophoresis is sodium dodecyl sulfonate-polyacrylamide gel electrophoresis
  • the polyacrylamide concentration is 12% (v/v)
  • the gel thickness is 0.75 mm
  • the voltage is 200 V
  • the electrophoresis time is 45 min.
  • alpha helix handle used to connect proteins and nucleic acids.
  • the specific position of the alpha helix handle contains unnatural amino acids
  • the unnatural amino acid is azidophenylalanine
  • the nucleic acid is connected with a coupling group, preferably a polyethylene glycol diphenyl cyclooctyne group;
  • the alpha helix handle and the protein to be tested are connected by a connecting polypeptide.
  • the present invention has the advantages and positive effects: the method uses the alpha helix handle to provide unnatural amino acids with controllable protein surface reaction conditions, avoids the complex structure, charge and polar nano-environment on the surface of the protein to be tested, thereby realizing the interaction between the protein and the protein. Nucleic acids are used for efficient click chemical ligation and other targets.
  • Figure 1 The operation flow of the method for improving the efficiency of protein and nucleic acid biological coupling based on the ⁇ -helix handle;
  • Figure 2 H-tag-PfAMA1-pET-21d recombinant expression plasmid map
  • Figure 3 The map of plasmid pEvol-pAzFRS.1.t1 carrying tRNA/aminoacyl tRNA synthetase;
  • Figure 4 Schematic diagram of the structure of the H-tag fusion protein in Example 1;
  • Figure 5 Three-dimensional structure diagram of the protein PfAMA1 to be tested in Example 1;
  • Figure 7 Schematic diagram of the tension-driven azide-alkynyl cyclization reaction in Example 1;
  • Figure 8 Sodium dodecylbenzene sulfonate-polyacrylamide gel electrophoresis chart in Example 1.
  • the principle of designing protein click chemistry coupling handles is to provide unnatural amino acids with the most reactive anchor point.
  • the secondary structure is the first element in designing protein coupling handles.
  • the alpha helix provides a stable coupling reaction anchor point and is the preferred handle structure. , Named H-tag.
  • the amino acid sequence of the alpha helix determines the local environment of the protein coupling reaction anchor point.
  • the ⁇ -helix handle is connected to the end of the protein by using a connecting peptide, and the unnatural amino acid is inserted into the specific position of the ⁇ -helix handle by using the extended genetic code. This is achieved through click chemistry. Efficient biological coupling of protein and nucleic acid.
  • the operation steps are:
  • Step 1 Based on the alpha helix of the protein secondary structure, design a handle H-tag carrying unnatural amino acids and connect it to one end of the protein to be tested through a polypeptide;
  • Step 2 Mutate a codon of the handle H-tag into an unnatural amino acid codon through site-specific genetics, construct a fusion recombinant expression plasmid containing the H-tag and the protein to be tested through a prokaryotic expression vector, and transfer it into competent cells to obtain the result after screening Stable genetic clone strain;
  • Step 3 Co-transform the fusion recombinant expression plasmid containing the H-tag and the protein to be tested and the plasmid capable of expressing tRNA/aminoacyl tRNA synthetase to obtain the prokaryotic expression strain, add unnatural amino acids to the medium, and induce the expression fusion with an inducer
  • the H-tag protein to be tested
  • Step 4 Lyse the cells of the prokaryotic expression strain after induced expression, and purify the soluble protein of H-tag-the protein to be tested with the aid of the affinity tag of the recombinant protein;
  • Step 5 Use click chemistry to efficiently connect the coupling group on the H-tag-test protein and the nucleic acid substrate, and use gel electrophoresis to check the reaction efficiency.
  • the unnatural amino acid is an unnatural amino acid coupled to an azide group, specifically azidophenylalanine.
  • click chemistry is a tension-driven azide-alkynyl cyclization reaction, and H-tag-the protein to be tested And nucleic acid substrate through azide group and diphenylcyclooctyne group to achieve rapid connection.
  • the length of the handle H-tag is greater than 5 amino acids and less than 30 amino acids. It can be connected to the amino or carboxyl end of the protein to be tested. Mutations in the alpha helix with less than 5 amino acids may damage the secondary structure of the alpha helix.
  • the linking peptide When its length exceeds 30 amino acids, its linking effect will be reduced; the linking peptide is longer than 8 amino acids and less than 20 amino acids. When the linking peptide is less than 8 amino acids, it will not be isolated from the protein surface amino acid environment. effect. And the preferred position of the unnatural amino acid in the H-tag sequence needs to meet the following conditions: neutrality, polar amino acid environment and moderate surface exposure.
  • the surface exposure can be obtained through the online software sable (http://sable.cchmc .org/), predict the relative solvent accessibility of the amino acid value, the total range is 0-5, in this scheme, the amino acid residue with the value 2 or 3 is selected as the candidate mutation site.
  • a codon of the handle H-tag is mutated into an unnatural amino acid codon.
  • the unnatural amino acid codon can be an amber codon or other codons that can realize the insertion function of an unnatural amino acid.
  • the principle of designing protein click chemistry coupling handles is to provide unnatural amino acids with the most reactive anchor point.
  • the secondary structure is the first element in designing protein coupling handles.
  • the alpha helix provides a stable coupling reaction anchor point and is the preferred handle structure. , Named H-tag, the amino acid sequence of the alpha helix determines the local environment of the protein coupling reaction anchor point.
  • Step 1 Based on the alpha helix of the protein secondary structure, design a handle H-tag carrying azidophenylalanine and connect it to one end of the protein to be tested through a polypeptide; design the H-tag as shown in the sequence SEQ ID NO:1 and connect The peptide fragment is shown in the sequence SEQ ID NO: 2;
  • nucleic acid sequence of the H-tag as shown in SEQ ID NO: 3, at the 5'end of the nucleic acid sequence of the protein to be tested, with a nucleic acid sequence encoding a connecting peptide segment in the middle, as shown in SEQ ID NO: 4 Connect to form a complete fusion protein.
  • Step 2 Mutation of a codon of the handle H-tag into an amber codon or other codons that can realize the function of unnatural amino acid insertion through site-specific genetics.
  • the structure is shown in Figure 4; for example, the H-tag amino acid sequence is encoded Site-directed genetic mutation of the 5th Gln codon to TAG, used to introduce azidophenylalanine, and then construct a fusion recombinant expression plasmid containing H-tag and the protein to be tested with the prokaryotic expression vector, and transfer it to competent cells Afterwards, screen cloned strains capable of stable inheritance;
  • the prokaryotic expression vector can be the pET series prokaryotic expression vector.
  • the competent cells of the cloned strain used for screening are E. coli DH5 ⁇ cells, or other E. coli strains that perform the same function; the method of constructing a recombinant expression plasmid can be through restriction nucleic acid After endonuclease digestion, use T4 ligase for ligation, or a ligation-independent cloning method (Ligation-independent cloning), such as Seamless Cloning.
  • Step 3 Extract the fusion recombinant expression plasmid containing the H-tag and the test protein from the cloned strain, and co-transform the fusion recombinant expression plasmid containing the H-tag and the test protein and the plasmid capable of expressing tRNA/aminoacyl tRNA synthetase.
  • Prokaryotic expression strain add unnatural amino acid, 4-azido-L-phenylalanine to the culture medium of prokaryotic expression strain, and use inducer to induce the expression of the test protein fused with H-tag;
  • the strain medium contains 5% glycerol and 1mM 4-azido-L-phenylalanine, the inducer uses 1mM IPTG, and the expression time is 12-20h; the orthogonal pair in the tRNA/aminoacyl tRNA synthetase plasmid
  • the coding gene can be single copy or multiple copies for the introduction of unnatural amino acids.
  • the plasmid used in this scheme is the plasmid pEvol-pAzFRS.1.t1 carrying tRNA/aminoacyl tRNA synthetase, purchased from the NTCC Type Culture Collection , Item No.: Plasmid 73547", the structure is shown in Figure 3.
  • Step 4 Lyse the cells of the prokaryotic expression strain after induced expression, and purify the soluble protein of H-tag-the protein to be tested with the aid of the affinity tag of the recombinant protein;
  • Protease inhibitors are preferably phenylmethylsulfonyl fluoride (PMSF), with a concentration of 0.1-1 mM, and other protease inhibitors can also be selected. Cell lysis is performed with an ultrasonic disruptor.
  • PMSF phenylmethylsulfonyl fluoride
  • the power is set to 200W, the cycle is on for 4 seconds and off for 6 seconds, and the total time is set to 25-40min;
  • the affinity tag can be hexahistidine, glutathione sulfhydryl transferase (GST), or other similar functions
  • GST glutathione sulfhydryl transferase
  • purified protein using chromatography technology, chromatography technology can choose affinity chromatography, such as metal chelation affinity chromatography, or a combination of ion exchange chromatography.
  • Step 5 Efficiently connect the purified H-tag-test protein to the coupling group on the nucleic acid substrate through the tension-driven azide-alkynyl cyclization reaction.
  • the reaction diagram is shown in Figure 7.
  • the coupling group on is a polyethylene glycol diphenyl cyclooctyne group, as shown in Figure 6, the molar ratio of the fusion protein to the nucleic acid substrate is 1:2.5-5, preferably 1:5, and the reaction temperature is 12°C, reaction time is 24h; use gel electrophoresis to check the reaction efficiency, gel electrophoresis is sodium dodecyl sulfonate-polyacrylamide gel electrophoresis, the concentration of polyacrylamide is 12% (v/v), The gel thickness is 0.75mm, the voltage is 200V, and the electrophoresis time is 45min.
  • This scheme has a certain degree of universality, and is especially suitable for soluble proteins and inclusion body proteins.
  • PfAMA1 protein as an example to describe the scheme of the present invention.
  • the experimental schemes not marked with specific conditions in the examples all follow the reaction conditions suggested in the conventional scheme or product specification.
  • the general equipment, materials and reagents in the examples can be purchased commercially unless otherwise specified.
  • the design of the H-tag sequence such as SEQ ID NO:1 provides moderate surface exposure, electrically neutral and polar amino acid environment for the unnatural amino acid azidophenylalanine in the protein coupling reaction.
  • Design a connecting peptide such as SEQ ID NO: 2, which is a linker or bridge to connect the protein to the H-tag (helix tag), which can isolate the target protein to be tested from the click chemistry coupling handle, and reduce the complex environment of the protein surface on the connection efficiency influences.
  • H-tag nucleic acid sequence such as SEQ ID NO: 3 at the 5'end of the nucleic acid sequence of the protein to be tested PfAMA1 (structure as shown in Figure 5), and connect it by a nucleic acid sequence encoding a connecting peptide in the middle to connect the peptide sequence Such as SEQ ID NO: 4, thus forming a complete fusion protein coding sequence, and its sequence is such as SEQ ID NO: 5.
  • the codon encoding Gln at position 5 of the H-tag amino acid sequence was site-directed genetically mutated to TAG (amber codon) for the introduction of azidophenylalanine.
  • the target sequence is obtained by polymerase chain reaction PCR; the upstream and downstream primers respectively include restriction nucleic acids Endonuclease sites NcoI and XhoI; in a 50 ⁇ l volume containing buffer (10mM Tris-HCl, (pH 8.5), 10mM MgCl 2 , 100mM KCl, 0.1mg/mL BSA), the target sequence (5 ⁇ g) and the large intestine Bacillus prokaryotic expression vector pET-21d (5 ⁇ g) was digested with NcoI (10U) and XhoI (10U) restriction endonucleases at 37°C for 1 hour, and separated by 1% agarose gel electrophoresis (120V, 20 minutes) , Gum cut recovery, purification to obtain the target sequence and vector containing sticky ends.
  • upstream primers such as SEQ ID NO: 6
  • downstream primers respectively include restriction nucleic acids Endonuclease sites NcoI and XhoI
  • a 10 ⁇ l volume containing buffer 40mM Tris-HCl, 10mM MgCl2, 10mM DTT, 0.5mM ATP
  • T4 DNA ligase (1U) to connect the target sequence (30ng) with the vector (51ng) to form a recombinant expression plasmid H -tag-PfAMA1-pET-21d.
  • the plasmid contains the gene encoding ampicillin, which can be used for screening positive clones.
  • a single positive clone was picked and inoculated in 5 ml LB medium containing 100 ⁇ g/ml ampicillin and 20 ⁇ g/ml chloramphenicol, and then cultured on a constant temperature shaker (220 revolutions/min) at 37° C. for 12 hours.
  • EDTA ethylenediaminetetraacetic acid
  • PMSF phenylmethylsulfonyl fluoride
  • Triton X-100 Triton X-100
  • the protein solution was subjected to anion exchange chromatography and cation exchange Chromatography and purification to obtain the target protein.
  • storage buffer (20mM Tris-HCl, pH 8.0, 200mM NaCl, 5% glycerol) at -80°C.
  • One of the substrates of the click chemical reaction is single-stranded ssDNA, as shown in SEQ ID NO: 8, synthesized by a commercial company, with a length of 14 deoxyribonucleotides, and a single polyethylene glycol diphenyl at its 3'end.
  • Dibenzocyclooctyne-polyethylene glycol (DBCO-PEG) (as shown in Figure 6). Remove the protein from the -80°C refrigerator and melt on ice. Mix protein and ssDNA in molar ratios of 1:5 and 1:2.5. When the molar ratio is 1:5, the final concentrations of protein and ssDNA are 40 ⁇ M and 200 ⁇ M, respectively.
  • the final concentrations of protein and ssDNA are respectively. It is 80 ⁇ M and 200 ⁇ M.
  • Add buffer ((20mM Tris-HCl, pH 8.0, 200mM NaCl), adjust the final reaction volume to 5 microliters. Pipette repeatedly 10 times until the mixture is uniform, and then react at 12°C for 25 hours. Pass dodecyl Sodium sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) identifies click chemistry reaction products.
  • the thickness of the 12% gel is 0.75 mm.
  • the voltage is 200 volts, the current is 400 mA, and the electrophoresis time is 40 minutes. It is bright in Coomassie.
  • H-tag is introduced into the amino end of the H-tag, and site-directed mutation of azidophenylalanine is performed on the H-tag.
  • the click chemistry yield of ssDNA is 50%.
  • the biological coupling efficiency of H-tag protein is increased by 40 %.

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Abstract

提供了一种基于α螺旋把手提高蛋白核酸生物耦合效率的方法。包括设计携带非天然氨基酸的把手H-tag，再构建含有H-tag和待测蛋白的融合重组表达质粒，表达纯化在H-tag中含有非天然氨基酸的融合重组蛋白，最后对融合蛋白H-tag中非天然氨基酸和核酸底物上的耦合基团进行高效点击化学连接。所述方法使用α螺旋把手为非天然氨基酸提供可控的蛋白表面反应条件，避免待测蛋白表面复杂的结构、电荷和极性纳米环境，从而实现对蛋白与核酸进行高效点击化学连接的目标。

Description

基于α螺旋把手提高蛋白核酸生物耦合效率的方法 技术领域
本发明属于药物化学生物学领域，尤其是涉及一种基于α螺旋把手提高蛋白核酸生物耦合效率的方法。
背景技术
目前生物耦合是研究生物大分子相互作用的重要工具，对于深刻理解大分子在细胞生命活动中的功能和药物在调节大分子生物活性的动态机制中具有重大意义，已经成为药物、化学和生物学交叉领域的前沿方向。生物耦合可以发生在蛋白与蛋白、蛋白与核酸、核酸与核酸、以及药物与蛋白核酸之间。常用的生物耦合反应有点击化学、二硫键和马来酰亚胺反应等。参与蛋白耦合的天然氨基酸的基团有氨基、羧基和巯基等。天然氨基酸的修饰基团位点多，总体反应活性高，易于进行生物耦合；但是缺点是位点选择特异性低，无法进行定点修饰。生物正交技术允许细胞使用翻译机器在蛋白中定点引入非天然氨基酸。这突破了天然氨基酸生物耦合基团的选择局限性，极大的丰富了修饰基团库。虽然蛋白的任一位置可通过定点遗传突变引入非天然氨基酸，但是蛋白表面的纳米环境复杂，导致难以预测非天然氨基酸耦合基团的反应活性。影响非天然氨基酸耦合基团反应活性的主要因素包括引入位点的溶剂暴露程度、氨基酸自身和周围电荷水平、以及临近氨基酸基团的极性特征等。通过非天然氨基酸进行蛋白耦合，需要经历复杂的突变位点优化选择程序，并且面临蛋白耦合效率低下的困境。此外，蛋白与核酸的生物耦合对于研究蛋白与核酸的相互作用至关重要。因此，开发一种携带非天然氨基酸引入位点的通用把手，用于对蛋白和核酸进行高效生物耦合，是非常必要的。
发明内容
为解决上述技术问题，本发明提供一种基于α螺旋把手提高蛋白核酸生物耦合效率的方法，用于优化设计蛋白耦合基团位置，特别是基于耦合反应的生物大分子相互作用探测。
本发明采用的技术方案是：基于α螺旋把手提高蛋白核酸生物耦合效率的方法，使用连接多肽将α螺旋把手与蛋白的末端进行连接，利用扩展遗传编码将非 天然氨基酸插入α螺旋把手的特定位点，通过点击化学，实现了蛋白与核酸的高效生物耦合。
具体步骤为：
步骤一基于蛋白二级结构α螺旋，设计携带非天然氨基酸的把手H-tag，通过多肽与待测蛋白的一个末端连接；
步骤二通过定点遗传将把手H-tag的一个密码子突变成非天然氨基酸密码子，通过原核表达载体构建含有H-tag和待测蛋白的融合重组表达质粒，转入感受态细胞后筛选获得稳定遗传的克隆菌株；
步骤三将含有H-tag和待测蛋白的融合重组表达质粒和能够表达tRNA/氨酰tRNA合成酶的质粒共转化获得原核表达菌株，在培养基中添加非天然氨基酸，使用诱导剂诱导表达融合了H-tag的待测蛋白；
步骤四裂解诱导表达后的原核表达菌株细胞，借助重组蛋白的亲和标签，纯化获得H-tag-待测蛋白的可溶蛋白；
步骤五通过点击化学对H-tag-待测蛋白和核酸底物上的耦合基团进行高效连接，使用凝胶电泳检测反应效率。
优选地，非天然氨基酸为非天然氨基酸耦合叠氮基团，具体为叠氮苯丙氨酸。
优选地，点击化学为张力驱动的叠氮-炔基环化反应。
优选地，步骤一中H-tag长度大于5个氨基酸，设计在待测蛋白的氨基端或羧基端；
优选地，连接多肽长度大于8个氨基酸；
优选地，非天然氨基酸在H-tag序列中的优选位置满足在电中性，极性氨基酸环境和氨基酸数值相对溶剂可及性为2-3的条件。
优选地，步骤二中原核表达载体为pET系列载体；
优选地，非天然氨基酸密码子为琥珀密码子；
优选地，含有H-tag和待测蛋白的融合重组表达质粒的构建能够通过依赖于连接的克隆方法或无缝克隆方法。
优选地，步骤三中培养基含有5％甘油和1mM非天然氨基酸；
优选地，诱导剂使用1mM异丙基-β-D-硫代半乳糖苷(IPTG)；
优选地，诱导表达时间优选12-20h，优选为16h；
优选地，能够表达tRNA/氨酰tRNA合成酶的质粒中的正交对编码基因可以是单拷贝或多拷贝。
优选地，步骤四中在蛋白酶抑制剂作用下裂解细胞，蛋白酶抑制剂优选苯甲基磺酰氟，浓度为0.1-1mM；
优选地，亲和标签为六聚组氨酸或谷胱甘肽巯基转移酶；
优选地，裂解细胞采用超声破碎仪，功率设置为200W，循环开4秒关6秒，总时间设置为25-40min；
优选地，使用层析技术纯化蛋白，优选为亲和层析，具体为金属螯合亲和层析或复合使用离子交换层析。
优选地，步骤五中核酸底物上的耦合基团为聚乙二醇二苯基环辛炔基团；
优选地，融合蛋白和核酸底物的摩尔比为1:2.5-5，优选为1:5；
优选地，反应温度为12℃，反应时间为24h；
优选地，凝胶电泳为十二烷基磺酸钠-聚丙烯酰胺凝胶电泳，聚丙烯酰胺的浓度为12％(v/v)，凝胶厚度为0.75mm，电压200V，电泳时间45min。
一种用于连接蛋白和核酸的α螺旋把手，α螺旋把手的特定位点含有非天然氨基酸；
优选地，所述非天然氨基酸为叠氮苯丙氨酸；
优选地，所述核酸连接有耦合基团，优选为聚乙二醇二苯基环辛炔基团；
优选地，所述α螺旋把手与待测蛋白通过连接多肽连接。
本发明具有的优点和积极效果是：该方法使用α螺旋把手为非天然氨基酸提供可控的蛋白表面反应条件，避免待测蛋白表面复杂的结构、电荷和极性纳米环境，从而实现对蛋白与核酸进行高效点击化学连接等目标。
附图说明
图1：基于α螺旋把手提高蛋白核酸生物耦合效率的方法操作流程；
图2：H-tag-PfAMA1-pET-21d重组表达质粒图谱；
图3：携带tRNA/氨酰tRNA合成酶的质粒pEvol-pAzFRS.1.t1图谱；
图4：实施例1中H-tag融合蛋白结构示意图；
图5：实施例1中待测蛋白PfAMA1三维结构图；
图6：实施例1中DBCO修饰的单链核酸结构；
图7：实施例1中张力驱动的叠氮-炔基环化反应示意图；
图8：实施例1中十二烷基苯磺酸钠-聚丙烯酰胺凝胶电泳图。
具体实施方式
下面结合附图对本发明的一个实施例做出说明。实施例中未标注具体条件的实验方案，皆按照常规方案或产品说明书中所建议的反应条件。实施例中的通用设备、材料和试剂等，如无特指，皆可从商业购买。
设计蛋白点击化学耦合把手的原则是为非天然氨基酸提供反应活性最高的锚点，二级结构是设计蛋白耦合把手的第一要素，α螺旋提供稳定的耦合反应锚点，是优先考虑的把手结构，命名为H-tag。α螺旋的氨基酸序列决定了蛋白耦合反应锚点的局域环境。
基于α螺旋把手提高蛋白核酸生物耦合效率的方法，使用连接多肽将α螺旋把手与蛋白的末端进行连接，利用扩展遗传编码将非天然氨基酸插入α螺旋把手的特定位点，通过点击化学，实现了蛋白与核酸的高效生物耦合。首先设计携带非天然氨基酸密码子的把手H-tag，其次构建含有H-tag和待测蛋白的融合重组表达质粒，再次表达纯化在H-tag中含有非天然氨基酸的融合重组蛋白，最后对融合蛋白H-tag中非天然氨基酸和核酸底物上的耦合基团进行高效点击化学连接，其流程如图1所示。
操作步骤为：
步骤一 基于蛋白二级结构α螺旋，设计携带非天然氨基酸的把手H-tag，通过多肽与待测蛋白的一个末端连接；
步骤二 通过定点遗传将把手H-tag的一个密码子突变成非天然氨基酸密码子，通过原核表达载体构建含有H-tag和待测蛋白的融合重组表达质粒，转入感受态细胞后筛选获得稳定遗传的克隆菌株；
步骤三 将含有H-tag和待测蛋白的融合重组表达质粒和能够表达tRNA/氨酰tRNA合成酶的质粒共转化获得原核表达菌株，在培养基中添加非天然氨基酸，使用诱导剂诱导表达融合了H-tag的待测蛋白；
步骤四 裂解诱导表达后的原核表达菌株细胞，借助重组蛋白的亲和标签，纯化获得H-tag-待测蛋白的可溶蛋白；
步骤五 通过点击化学对H-tag-待测蛋白和核酸底物上的耦合基团进行高效 连接，使用凝胶电泳检测反应效率。
其中，非天然氨基酸为非天然氨基酸耦合叠氮基团，具体可为叠氮苯丙氨酸，相应的，点击化学为张力驱动的叠氮-炔基环化反应，H-tag-待测蛋白和核酸底物通过叠氮基团和二苯基环辛炔基团实现快速连接。把手H-tag长度大于5个氨基酸，并且小于30个氨基酸，可以连接在待测蛋白的氨基端或羧基端，少于5个氨基酸在α螺旋上做突变可能会破坏α螺旋这种二级结构，当其长度超过30个氨基酸后，其连接效果会随之降低；连接多肽长度大于8个氨基酸，小于20个氨基酸，当连接多肽少于8个氨基酸会起不到与蛋白表面氨基酸环境的隔离作用。并且非天然氨基酸在H-tag序列中的优选位置，需要满足如下条件，电中性、极性氨基酸环境和适中的表面暴露度，表面暴露度可通过在线软件sable(http://sable.cchmc.org/)，预测氨基酸的数值相对溶剂可及性，总范围是0-5，本方案中选择数值是2或3的氨基酸残基作为候选突变位点。通过定点遗传将把手H-tag的一个密码子突变成非天然氨基酸密码子，非天然氨基酸密码子可以是琥珀密码子，也可以是能实现非天然氨基酸插入功能的其它密码子。
设计蛋白点击化学耦合把手的原则是为非天然氨基酸提供反应活性最高的锚点，二级结构是设计蛋白耦合把手的第一要素，α螺旋提供稳定的耦合反应锚点，是优先考虑的把手结构，命名为H-tag，α螺旋的氨基酸序列决定了蛋白耦合反应锚点的局域环境。
具体操作步骤如下：
步骤一 基于蛋白二级结构α螺旋，设计携带叠氮苯丙氨酸的把手H-tag，通过多肽与待测蛋白的一个末端连接；设计H-tag如序列SEQ ID NO:1所示，连接肽段如序列SEQ ID NO:2所示；
SEQ ID NO:1 DITQQAKDIG
SEQ ID NO:2 GSGGGSGG
将H-tag的核酸序列，如SEQ ID NO:3所示，置于待测蛋白核酸序列的5’端，中间通过编码一个连接肽段的核酸序列，如SEQ ID NO:4所示，进行连接，从而形成一个完整的融合蛋白。
SEQ ID NO:3 GATATAACACAACAAGCTAAAGATATAGGC
SEQ ID NO:4 GGTAGCGGTGGCGGAAGCGGCGGT
步骤二 通过定点遗传将把手H-tag的一个密码子突变成琥珀密码子或者是能实现非天然氨基酸插入功能的其它密码子，其结构如图4所示；例如将编码H-tag氨基酸序列的第5位Gln的密码子定点遗传突变为TAG，用于引入叠氮苯丙氨酸，再与原核表达载体构建含有H-tag和待测蛋白的融合重组表达质粒，转入到感受态细胞后，筛选能够稳定遗传的克隆菌株；
其中原核表达载体可选用pET系列原核表达载体，用于筛选的克隆菌株的感受态细胞选用大肠杆菌DH5α细胞，或执行同样功能的其它大肠杆菌菌株；构建重组表达质粒的方法可以是通过限制性核酸内切酶消化后使用T4连接酶连接，也可以是不依赖于连接的克隆方法(Ligation-independent cloning)，譬如无缝克隆(Seamless Cloning)。
步骤三 从克隆菌株中提取含有H-tag和待测蛋白的融合重组表达质粒，将含有H-tag和待测蛋白的融合重组表达质粒和能够表达tRNA/氨酰tRNA合成酶的质粒共转化获得原核表达菌株，在原核表达菌株培养基中添加非天然氨基酸，4-叠氮基-L-苯丙氨酸，使用诱导剂诱导表达融合了H-tag的待测蛋白；
其中，菌株培养基含有5％甘油和1mM 4-叠氮基-L-苯丙氨酸，诱导剂使用1mM IPTG，诱导表达时间12-20h；tRNA/氨酰tRNA合成酶质粒中的正交对编码基因可以是单拷贝或多拷贝，用于引入非天然氨基酸，本方案使用的质粒是携带tRNA/氨酰tRNA合成酶的质粒pEvol-pAzFRS.1.t1，购自“NTCC典型培养物保藏中心，货号：Plasmid 73547”，结构如图3所示。
步骤四 裂解诱导表达后的原核表达菌株细胞，借助重组蛋白的亲和标签，纯化获得H-tag-待测蛋白的可溶蛋白；
细胞的裂解在蛋白酶抑制剂的作用下进行，蛋白酶抑制剂优选苯甲基磺酰氟(phenylmethylsulfonyl fluoride，PMSF)，浓度为0.1-1mM，也可选择其它蛋白酶抑制剂；裂解细胞采用超声破碎仪，功率设置为200W，循环开4秒关6秒，总时间设置为25-40min；亲和标签可以选择六聚组氨酸，也可以选择谷胱甘肽巯基转移酶(GST)，或其它类似功能的标签；纯化蛋白使用层析技术，层析技术可以选择亲和层析，譬如金属螯合亲和层析，也可以复合使用离子交换层析。
步骤五 通过张力驱动的叠氮-炔基环化反应对纯化所得的H-tag-待测蛋白和核酸底物上的耦合基团进行高效连接，反应示意图如图7所示，其核酸底物上的 耦合基团为聚乙二醇二苯基环辛炔基团，如图6所示，融合蛋白和核酸底物的摩尔比为1:2.5-5，优选为1:5，反应温度为12℃，反应时间为24h；使用凝胶电泳检测反应效率，凝胶电泳为十二烷基磺酸钠-聚丙烯酰胺凝胶电泳，聚丙烯酰胺的浓度为12％(v/v)，凝胶厚度为0.75mm，电压200V，电泳时间45min。
本方案具有一定的普适性，尤其适用于可溶性蛋白和包涵体蛋白，下面以PfAMA1蛋白为例详述本发明方案。实施例中未标注具体条件的实验方案，皆按照常规方案或产品说明书中所建议的反应条件。实施例中的通用设备、材料和试剂等，如无特指，皆可从商业购买。
实施例：H-tag应用于PfAMA1蛋白与单链核酸的高效生物耦合
1 H-tag的设计
设计H-tag序列如SEQ ID NO:1，为蛋白耦合反应中非天然氨基酸叠氮苯丙氨酸提供适中的表面暴露度，电中性和极性氨基酸环境。设计连接肽段如序列SEQ ID NO:2，为linker或桥梁将蛋白与H-tag(螺旋标签)连接，能够将待测目的蛋白与点击化学耦合把手隔离，降低蛋白表面复杂环境对连接效率的影响。
SEQ ID NO:1 DITQQAKDIG
SEQ ID NO:2 GSGGGSGG
将H-tag的核酸序列如SEQ ID NO:3，置于待测蛋白PfAMA1(结构如图5)的核酸序列的5’端，中间通过编码一个连接肽段的核酸序列连接，连接肽段序列如SEQ ID NO:4，从而形成一个完整的融合蛋白编码序列，其序列如SEQ ID NO:5。其中，将编码H-tag氨基酸序列的第5位Gln的密码子定点遗传突变为TAG(琥珀密码子)，用于引入叠氮苯丙氨酸。
SEQ ID NO:3 GATATAACACAACAAGCTAAAGATATAGGC
SEQ ID NO:4 GGTAGCGGTGGCGGAAGCGGCGGT
SEQ ID NO:5
Figure PCTCN2019107289-appb-000001
Figure PCTCN2019107289-appb-000002
2氨基端带有H-tag序列的PfAMA1融合重组蛋白表达载体的构建
以融合蛋白编码序列为模板，使用上游引物(如SEQ ID NO:6)和下游引物(如SEQ ID NO:7)，通过聚合酶链式反应PCR获得目的序列；上下游引物分别包括限制性核酸内切酶位点NcoI和XhoI；在含有缓冲液(10mM Tris-HCl，(pH 8.5),10mM MgCl 2,100mM KCl,0.1mg/mL BSA)的50μl体积中，将目的序列(5μg)和大肠杆菌原核表达载体pET-21d(5μg)使用NcoI(10U)和XhoI(10U)限制性核酸内切酶在37℃酶切消化1小时，经1％琼脂糖凝胶电泳(120V，20分钟)分离，切胶回收，纯化获得含有粘性末端的目的序列和载体。在含有缓冲液(40mM Tris-HCl,10mM MgCl2,10mM DTT,0.5mM ATP)的10μl体积中，利用T4 DNA连接酶(1U)将目的序列(30ng)与载体(51ng)连接构成重组表达质粒H-tag-PfAMA1-pET-21d。该质粒含有氨苄青霉素编码基因，可用于阳性克隆的筛选。
Figure PCTCN2019107289-appb-000003
3重组蛋白H-tag-PfAMA1的表达
将重组表达质粒H-tag-PfAMA1-pET-21d(如图2所示)与携带tRNA/氨酰tRNA合成酶的质粒pEvol-pAzFRS.1.t1(如图3所示)共转大肠杆菌感受态细胞 BL21(DE3)，转化方法参考《分子克隆实验指南(第三版)》(科学出版社2002，黄培堂等译)。挑取单个阳性克隆，接种于含100μg/ml氨苄青霉素和20μg/ml氯霉素的5ml LB培养基，继而在37℃恒温摇床震荡(220转/分钟)培养12小时。将菌液转接至含100μg/ml氨苄青霉素和20μg/ml氯霉素的1L LB培养基，在37℃恒温摇床(220转/分钟)继续培养，当菌液光密度OD 600达到0.6-0.8时加入终浓度为1mM IPTG、1mM叠氮苯丙氨酸(4-azido-L-Phenoalanine)和5％甘油，在37℃恒温摇床(220转/分钟)诱导表达16小时。4000转/分钟离心收集细胞。用裂解缓冲液(20mM Tris-HCl,pH 8.0,400mM NaCl,5％甘油)重悬细胞，细胞悬液可直接进行下一步纯化实验或于-80℃保存。
4重组蛋白H-tag-PfAMA1的纯化
向步骤3中的细胞悬液中加入终浓度为2mM的乙二胺四乙酸(EDTA)、0.2mM苯甲基磺酰氟(PMSF)、0.1％曲拉通X-100(Triton X-100)，并定容至100ml。使用超声破碎仪(宁波新芝生物科技股份有限公司，型号：SCIENTZ-IID)功率为200W，循环开4秒关6秒，总时间40分钟，充分裂解细胞。细胞裂解液于18000转/分离心40分钟，弃去上清液，保留沉淀的蛋白质。用40ml清洗缓冲液(20mM Tris-HCl,pH 8.0,2M尿素,0.5mM EDTA)清洗两次沉淀蛋白质。用50ml溶解缓冲液(20mM Tris-HCl,pH 8.0,6M盐酸胍(Guanidine hydrochloride，GdnHCl),2mM 2-巯基乙醇(2-Mercaptoethanol，2-ME)溶解沉淀的蛋白质12小时。将溶解液于18000转/分离心40分钟，收集上清液中溶解的蛋白质，弃去沉淀。用缓冲液(20mM Tris-HCl,pH 8.0,6M GdnHCl)将蛋白溶液调整浓度为20μg/ml，并定容至50ml。将盛有蛋白溶液的透析袋放置于3L复性缓冲液(20mM Tris–HCl,pH 8.0,0.5mM EDTA,1M尿素,200mM NaCl,2mM 2-ME,0.2mM胱胺二盐酸盐(Cystamine–HCl))中，于4℃搅拌透析12小时，进行复性。复性完成后将蛋白溶液转移至3L裂解缓冲液，于4℃搅拌透析12小时。18000转/分离心40分钟除去因错误折叠而析出的蛋白沉淀。取上清，过3ml Ni亲和层析柱，4℃孵育3小时。用含20mM咪唑的裂解缓冲液洗去杂蛋白。用含500mM咪唑裂解缓冲液洗脱目的蛋白。将洗脱的蛋白溶液透析至3L阴离子交换层析结合缓冲液(20mM Tris-HCl,pH 8.0,40mM NaCl)，于4℃搅拌透析12小时。随后，蛋白质溶液经过阴离子交换层析和阳离子交换层析， 纯化获得目的蛋白。在存储缓冲液(20mM Tris-HCl,pH 8.0,200mM NaCl,5％甘油)中冻存于-80℃。
5 H-tag-PfAMA1与ssDNA的点击化学耦合
点击化学反应底物之一是单链ssDNA，如SEQ ID NO:8所示，经商业公司合成，长度为14个脱氧核糖核苷酸，其3’末端修饰有单个聚乙二醇二苯基环辛炔基团(Dibenzocyclooctyne-polyethylene glycol,DBCO-PEG)(如图6所示)。从-80℃冰箱取出蛋白质，冰上融化。分别以1:5和1:2.5的摩尔比例混合蛋白与ssDNA，摩尔比例为1:5时，蛋白与ssDNA终浓度分别为40μM与200μM，摩尔比例为1:2.5时，蛋白与ssDNA终浓度分别为80μM与200μM。添加缓冲液((20mM Tris-HCl,pH 8.0,200mM NaCl)，调节最终反应体积至5微升。移液器反复吹打10次直至混合均匀，然后在12℃反应25小时。通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定点击化学反应产物。12％凝胶的厚度是0.75毫米。电压是200伏特，电流是400毫安，电泳时间40分钟。在考马斯亮蓝R-250染色液(0.1％(w/v)考马斯亮蓝R-250，25％异丙醇(v/v)，10％冰醋酸(v/v))中染色10分钟，在考马斯亮蓝染色脱色液(10％(v/v)醋酸，5％(v/v)乙醇)中脱色，脱色时间以能看到清晰蛋白条带，基本无背景色为准。使用全能型成像系统(SYNGENE，型号：PXi903030611)进行拍照。如图8所示，在PfAMA1内部选择并引入叠氮苯丙氨酸突变位点的蛋白底物，与ssDNA进行点击化学反应，产率是10％。在PfAMA1的氨基端引入H-tag，并在H-tag上进行叠氮苯丙氨酸定点突变，与ssDNA的点击化学产率是50％，两相对比，H-tag蛋白的生物耦合效率提高了40％。
SEQ ID NO:8 CGTCTGACCGTAAC
以上对本发明的一个实施例进行了详细说明，但所述内容仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等，均应仍归属于本发明的专利涵盖范围之内。
Figure PCTCN2019107289-appb-000004
Figure PCTCN2019107289-appb-000005
Figure PCTCN2019107289-appb-000006
Figure PCTCN2019107289-appb-000007

Claims (10)

  1. 基于α螺旋把手提高蛋白核酸生物耦合效率的方法，其特征在于：连接多肽将α螺旋把手与蛋白末端连接，利用扩展遗传编码将非天然氨基酸插入α螺旋把手特定位点，通过点击化学，实现蛋白与核酸的高效生物耦合。
  2. 根据权利要求1所述的基于α螺旋把手提高蛋白核酸生物耦合效率的方法，其特征在于：具体步骤为：
    步骤一 基于蛋白二级结构α螺旋，设计把手H-tag，通过多肽与待测蛋白的末端连接；
    步骤二 通过定点遗传将把手H-tag的一个密码子突变成非天然氨基酸密码子，通过原核表达载体构建含有H-tag和待测蛋白的融合重组表达质粒，转入感受态细胞后筛选获得稳定遗传的克隆菌株；
    步骤三 将含有H-tag和待测蛋白的融合重组表达质粒和能够表达tRNA/氨酰tRNA合成酶的质粒共转化获得原核表达菌株，在培养基中添加所述非天然氨基酸，使用诱导剂诱导表达融合了H-tag的待测蛋白；
    步骤四 裂解诱导表达后的原核表达菌株细胞，借助重组蛋白的亲和标签，纯化获得H-tag-待测蛋白的可溶蛋白；
    步骤五 通过点击化学对H-tag-待测蛋白和核酸底物上的耦合基团进行高效连接，使用凝胶电泳检测反应效率。
  3. 根据权利要求1或2所述的基于α螺旋把手提高蛋白核酸生物耦合效率的方法，其特征在于：所述非天然氨基酸为非天然氨基酸耦合叠氮基团，具体为叠氮苯丙氨酸。
  4. 根据权利要求3所述的基于α螺旋把手提高蛋白核酸生物耦合效率的方法，其特征在于：所述点击化学为张力驱动的叠氮-炔基环化反应。
  5. 根据权利要求3或4所述的基于α螺旋把手提高蛋白核酸生物耦合效率的方法，其特征在于：步骤一中所述H-tag长度大于5个氨基酸，设计在待测蛋白的氨基端或羧基端；
    优选地，所述连接多肽长度大于8个氨基酸；
    优选地，非天然氨基酸在H-tag序列中的位置满足在电中性，极性氨基酸环境和氨基酸数值相对溶剂可及性为2-3的条件。
  6. 根据权利要求3或4所述的基于α螺旋把手提高蛋白核酸生物耦合效率的方法， 其特征在于：所述步骤二中原核表达载体为pET系列载体；
    优选地，非天然氨基酸密码子为琥珀密码子；
    优选地，含有H-tag和待测蛋白的融合重组表达质粒的构建能够通过依赖于连接的克隆方法或无缝克隆方法。
  7. 根据权利要求3或4所述的基于α螺旋把手提高蛋白核酸生物耦合效率的方法，其特征在于：所述步骤三中培养基含有5％甘油和1mM非天然氨基酸；
    优选地，诱导剂使用1mM异丙基-β-D-硫代半乳糖苷(IPTG)；
    优选地，诱导表达时间优选12-20h，优选为16h；
    优选地，能够表达tRNA/氨酰tRNA合成酶的质粒中的正交对编码基因是单拷贝或多拷贝。
  8. 根据权利要求3或4所述的基于α螺旋把手提高蛋白核酸生物耦合效率的方法，其特征在于：所述步骤四中在蛋白酶抑制剂作用下裂解细胞，所述蛋白酶抑制剂优选苯甲基磺酰氟，浓度为0.1-1mM；
    优选地，所述亲和标签为六聚组氨酸或谷胱甘肽巯基转移酶；
    优选地，裂解细胞采用超声破碎仪，功率设置为200W，循环开4秒关6秒，总时间设置为25-40min；
    优选地，使用层析技术纯化蛋白，优选为亲和层析，具体为金属螯合亲和层析或复合使用离子交换层析。
  9. 根据权利要求3或4所述的基于α螺旋把手提高蛋白核酸生物耦合效率的方法，其特征在于：所述步骤五中核酸底物上的耦合基团为聚乙二醇二苯基环辛炔基团；
    优选地，融合蛋白和核酸底物的摩尔比为1:2.5-5，优选为1:5；
    优选地，反应温度为12℃，反应时间为24h；
    优选地，凝胶电泳为十二烷基磺酸钠-聚丙烯酰胺凝胶电泳，聚丙烯酰胺的浓度为12％(v/v)，凝胶厚度为0.75mm，电压200V，电泳时间45min。
  10. 一种用于连接蛋白和核酸的α螺旋把手，其特征在于：α螺旋把手的特定位点含有非天然氨基酸；
    优选地，所述非天然氨基酸为叠氮苯丙氨酸；
    优选地，所述核酸连接有耦合基团，优选为聚乙二醇二苯基环辛炔基团；
    优选地，所述α螺旋把手与待测蛋白通过连接多肽连接。
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