Streptavidin mutants and their uses

Abstract

This application discloses a streptavidin mutant and its uses. Compared to wild-type streptavidin, the streptavidin mutant contains a mutation, namely G48A / M / P / H / L, and the amino acid sequence of the wild-type streptavidin is shown in SEQ ID NO:1. The streptavidin mutant of this application has advantages such as high thermostability and strong biotin-binding activity. In particular, compared with wild-type streptavidin, the Tm value of the streptavidin mutant of this application is significantly increased, and it also exhibits superior biotin affinity (especially HABA binding ability) compared to wild-type streptavidin.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a streptavidin mutant and its uses. Background Technology

[0002] Streptavidin is a tetrameric protein derived from the actinomycete *Streptomyces avidinii*, with each subunit having a molecular weight of approximately 13 kDa. It forms a highly affinity, non-covalently bound to biotin (vitamin H or B7), with a dissociation constant reaching 10⁻¹⁴-10⁻¹⁵ M. Compared to avidin derived from egg white, streptavidin lacks glycosylation, thus exhibiting a lower non-specific binding background, making it more suitable for biological detection and analysis applications. Due to the extremely high binding affinity and specificity between streptavidin and biotin, it is widely used in molecular biology, medical diagnostics, and materials science. In biological detection, streptavidin is often bound to biotinylated antibodies, nucleic acid probes, or proteins for experiments such as enzyme-linked immunosorbent assays (ELISA), Western blot, immunohistochemistry, and flow cytometry, significantly improving detection sensitivity and specificity. In the field of molecular purification, the binding of streptavidin to biotin enables the efficient separation and immobilization of target molecules, commonly used in affinity chromatography, magnetic bead separation, and microarray immobilization. In nucleic acid analysis, the streptavidin-biotin system is used for nucleic acid hybridization signal amplification, biological sequencing, and microarray detection, improving signal stability and detection reliability. In drug delivery and targeted therapy, streptavidin can serve as a bridge, precisely guiding biotinylated drugs, antibodies, or nanocarriers to target tissues or cells. Furthermore, the development of monomeric streptavidin, fusion proteins, or truncated streptavidin through genetic engineering further expands its application potential in diagnostics, therapy, and the development of novel biomaterials.

[0003] Wild-type streptavidin exhibits excellent binding performance at room temperature, but its molecular stability has certain limitations. In practical applications, many bioprocesses and detection technologies often involve non-physiological temperature conditions. For example, in the thermal cycling process of polymerase chain reaction (PCR), certain high-temperature enzymatic reactions, and harsh elution processes based on streptavidin magnetic beads, the reaction system may be exposed to high temperatures (e.g., above 70°C) for short periods or continuously. Under such high-temperature environments, wild-type streptavidin is prone to conformational changes or thermal denaturation, leading to a significant decrease in its binding ability to biotin, thereby affecting detection sensitivity and purification efficiency.

[0004] To improve the stability of streptavidin, current research mainly focuses on modifying it through genetic engineering. However, while most reported mutation strategies can increase the protein's melting temperature (Tm) to some extent, they often impair biotin affinity, resulting in lower capture efficiency of mutants compared to the wild type in practical applications. Therefore, how to enhance the thermal stability of streptavidin while maintaining biotin-binding activity comparable to the wild type has become a technical bottleneck for expanding the application of this system under high-temperature conditions. Summary of the Invention

[0005] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a streptavidin mutant.

[0006] This application is based on the following discoveries of the inventors: Currently, wild-type streptavidin and its common mutants suffer from the following technical limitations when applied in molecular diagnostics, biosensors, and separation and purification: 1. Insufficient thermal stability, making it difficult to adapt to extreme reaction conditions: While existing wild-type streptavidin exhibits good binding properties at room temperature, its thermal stability is poor. m The value is relatively low. In applications such as molecular diagnostics that require high-temperature denaturation (e.g., PCR amplification) or long-term storage, existing proteins are prone to structural delamination or inactivation due to heat, making them unable to directly participate in reactions at high temperatures or maintain long-term function under harsh conditions, thus limiting their potential for use in processes involving high temperatures.

[0007] 2. The challenge of balancing stability and activity: Existing technologies for modifying streptavidin often present a trade-off: while certain mutation methods can slightly improve protein stability, they often come at the cost of sacrificing biotin-binding activity, leading to a decrease in the mutant's affinity. This makes it difficult for current technologies to provide high-performance mutants that are both heat-resistant and maintain high affinity, significantly reducing their practicality.

[0008] 3. There is a scarcity of available mutants, and a lack of gradient selection for different temperature ranges: Current technologies offer a limited variety of streptavidin mutants for screening, and lack systematic exploration of different mutation sites. This makes it difficult for researchers to find candidate protein libraries with gradient thermal stability when facing different application scenarios (such as processes requiring different temperature tolerances), and they cannot flexibly select the optimal mutant according to specific temperature requirements, thus limiting the potential for further improvement in detection sensitivity or purification efficiency.

[0009] Based on this, this application screened amino acid site G48, the hydrophobic core, subunit interface, and key flexible loop region of wild-type streptavidin, using tetrameric three-dimensional structure analysis. Through site-directed mutagenesis, the amino acid at this site was replaced with an amino acid that has higher hydrophobicity and is more likely to form hydrogen bonds or salt bridges. The melting temperature (Tm) of the mutant was then detected using the Thermo Fisher Scientific Protein Thermal Shift™ Dye Kit and StepOnePlus™ real-time quantitative PCR system. After obtaining mutants with high Tm values, their biotin-binding activity was detected using a HABA (2-hydroxyazobenzoic acid-4'-carboxylic acid) competitive binding assay. The results showed that the enzyme activity of the streptavidin mutant in this application was slightly higher than that of wild-type streptavidin. Furthermore, using a biotin-to-HABA method based on SA magnetic beads, the change in free HABA at 350 nm was measured, further revealing that the streptavidin mutant exhibited superior HABA-binding and biotin-binding abilities compared to wild-type streptavidin under both heated and unheated conditions.

[0010] Therefore, in a first aspect of this application, a streptavidin mutant is proposed. According to an embodiment of this application, the streptavidin mutant has a mutation compared to wild-type streptavidin, the mutation being G48A / M / P / H / L; the amino acid sequence of the wild-type streptavidin is shown in SEQ ID NO:1.

[0011] According to embodiments of this application, compared to wild-type streptavidin, the streptavidin mutant further lacks amino acids from position 2 to position 12.

[0012] According to embodiments of this application, the amino acid sequence of the streptavidin mutant is shown in any one of SEQ ID NO:2~6.

[0013] In a second aspect of this application, this application proposes a method to improve the stability of streptavidin or T m A method for determining the value. According to embodiments of this application, the method includes: mutating the 48th amino acid of wild-type streptavidin to increase streptavidin T. m The mutation is G48A / M / P / H / L, and the amino acid sequence of the wild-type streptavidin is shown in SEQ ID NO:1.

[0014] According to an embodiment of this application, the method further includes: deleting amino acids 2 to 12 of wild-type streptavidin.

[0015] In a third aspect of this application, a biological material is proposed, comprising any one of the following (i) to (iii): (i) a nucleic acid molecule that encodes the streptavidin mutant described in the first aspect; (ii) a vector carrying the nucleic acid molecule described in (i); (iii) Recombinant cells comprising the nucleic acid molecule described in (i) or the vector described in (ii), or expressing the streptavidin mutant described in the first aspect.

[0016] In a fourth aspect of this application, a complex is proposed. According to an embodiment of this application, the complex comprises the streptavidin mutant described in the first aspect and a streptavidin conjugate; the streptavidin mutant and the streptavidin conjugate are linked together.

[0017] According to embodiments of this application, the streptavidin conjugate is selected from at least one of biotin or biotin-conjugated molecules, molecular probes, markers, and solid-phase supports.

[0018] In a fifth aspect of this application, a reagent or kit is provided. According to embodiments of this application, the reagent or kit comprises the streptavidin mutant described in the first aspect, the biomaterial described in the third aspect, or the complex described in the fourth aspect.

[0019] In a sixth aspect of this application, the use of the streptavidin mutant described in the first aspect, the biomaterial described in the third aspect, the complex described in the fourth aspect, or the reagent or kit described in the fifth aspect in the preparation of products for biological detection, molecular purification, nucleic acid analysis, drug delivery, and targeted drug therapy is proposed.

[0020] Beneficial effects: The streptavidin mutant of this application has advantages such as high thermal stability and strong biotin-binding activity. In particular, compared with wild-type streptavidin, the streptavidin mutant T of this application... m The value was significantly improved, and it also showed superior biotin affinity (especially HABA binding ability) compared to wild-type streptavidin.

[0021] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0022] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 The images show SDS-PAGE electrophoresis diagrams (left) and (right) of the SA-G48A mutant and SA-G48M mutant in Example 2 of this application. Figure 2 The results of Hiload 16 / 600 superdex 75 pg molecular sieve analysis for the SA-G48A mutant in Example 2 of this application; Figure 3 The results of Hiload 16 / 600 superdex 75 pg molecular sieve analysis for the SA-G48M mutant in Example 2 of this application; Figure 4 The StepOnePlus™ system in Example 3 of this application was used to detect the SA-G48A mutant T. m Value result; Figure 5 The results of the StepOnePlus™ system detecting the Tm value of the SA-G48M mutant in Example 3 of this application; Figure 6 The streptavidin mutant and wild-type streptavidin T in Example 4 of this application m Value comparison chart; Figure 7 The results are from the biotin titration test of streptavidin mutant and wild-type streptavidin in Example 5 of this application. Detailed Implementation

[0023] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0024] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0025] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0026] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0027] In this document, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule that contains one or more nucleotide or amino acid differences (mutations) that differ from a reference sequence. This difference can be a substitution, deletion, or insertion of one or more amino acids.

[0028] In this article, the term "T" m Melting temperature refers to the temperature at which half of the protein molecules undergo irreversible denaturation during the thermal denaturation process, when the native folded conformation and the denatured extended conformation reach thermodynamic equilibrium. m It is a key quantitative indicator for measuring the thermal stability of proteins: T m A higher T value indicates a stronger resistance to heat denaturation and higher conformational stability of the protein; conversely, a lower T value indicates a lower protein stability. m The lower the value, the weaker the thermal stability.

[0029] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0030] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of this application and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0031] This application proposes a streptavidin mutant and its uses, which will be described in detail below.

[0032] Streptavidin mutant In a first aspect of this application, a streptavidin mutant is proposed. According to an embodiment of this application, the streptavidin mutant has a mutation compared to wild-type streptavidin, the mutation being G48A / M / P / H / L, and the amino acid sequence of the wild-type streptavidin is shown in SEQ ID NO:1.

[0033] The streptavidin mutant of this application, compared to the wild-type streptavidin T... m The value increased significantly, T m The increased biotin binding activity indicates that the streptavidin mutant of this application exhibits stronger structural stability under high-temperature conditions, making it suitable for applications in high-temperature environments. Furthermore, the streptavidin mutant of this application demonstrates superior biotin affinity (especially HABA binding ability) compared to wild-type streptavidin, resolving the common technical contradiction in this field of "increased stability but decreased activity," thus ensuring its practical value and enabling the development of molecular diagnostic tools, biosensors, or separation and purification materials suitable for different temperature requirements. Therefore, the streptavidin mutant of this application possesses advantages such as high thermal stability and strong biotin binding activity.

[0034] Unless otherwise specified in this article, all mutation sites are obtained by encoding the full-length sequence of wild-type streptavidin from the N-terminus to the C-terminus, where the full-length sequence of wild-type streptavidin is shown in SEQ ID NO:1.

[0035] MDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 1).

[0036] According to embodiments of this application, the above-mentioned streptavidin mutant may further include at least one of the following technical features: According to embodiments of this application, compared to wild-type streptavidin, the streptavidin mutant further has missing amino acids.

[0037] According to an embodiment of this application, the missing amino acid is located at the N-terminus.

[0038] According to embodiments of this application, the number of missing amino acids is 2 to 15, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or any range between any two of them.

[0039] According to embodiments of this application, compared to wild-type streptavidin, the streptavidin mutant further lacks amino acids from position 2 to position 12.

[0040] In this study, the amino acids at positions 2 through 12 of wild-type streptavidin are DPSKESKAQAAV (SEQ ID NO: 8).

[0041] According to embodiments of this application, the streptavidin mutant has an amino acid sequence as shown in any one of SEQ ID NO:2-6 or has a conserved amino acid substitution thereon.

[0042] In this document, "substitution of conserved amino acids" does not significantly affect or alter the binding properties of conjugates containing that amino acid sequence. Substitution can be introduced into the conjugates of this application using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved amino acid substitution refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the conjugates of this application may be replaced by other amino acid residues from the same side chain family, and the retained function of the modified conjugates may be tested using the functional assay methods described herein. Preferably, the number of conservative modifications does not exceed one, two, three, four, or five.

[0043] According to embodiments of this application, the streptavidin mutant has an amino acid sequence as shown in any one of SEQ ID NO:2-6.

[0044] According to embodiments of this application, the amino acid sequence of the streptavidin mutant is shown in any one of SEQ ID NO:2~6.

[0045] Unless otherwise specified herein, "amino acid sequence as shown in SEQ ID NO:A" includes the amino acid sequence shown in SEQ ID NO:A, and also includes amino acid sequences with conserved amino acid substitutions based on the amino acid sequence shown in SEQ ID NO:A. For example, "amino acid sequence as shown in SEQ ID NO:2" includes the amino acid sequence shown in SEQ ID NO:2, and also includes amino acid sequences with conserved amino acid substitutions based on the amino acid sequence shown in SEQ ID NO:2.

[0046] G48A mutant: MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVANAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO:2); G48M mutant: MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVMNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO:3); G48P mutant: MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVPNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO:4); G48H mutant: MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVHNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO:5); G48L mutant: MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVLNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 6).

[0047] According to an embodiment of this application, the truncated sequence of the wild-type streptavidin is shown in SEQ ID NO:7.

[0048] MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO: 7).

[0049] Improve streptavidin stability or T m Value method In a second aspect of this application, this application proposes a method to improve the stability of streptavidin or T m A method for determining the value. According to embodiments of this application, the method includes: mutating the 48th amino acid of wild-type streptavidin to increase streptavidin T. m The mutation is G48A / M / P / H / L, and the amino acid sequence of the wild-type streptavidin is shown in SEQ ID NO:1.

[0050] The inventors unexpectedly discovered that by mutating the 48th amino acid of wild-type streptavidin, its T... m This enhances its structural stability under high-temperature conditions, making it suitable for applications in high-temperature environments. Furthermore, it retains its biotin affinity (especially its binding ability to HABA), resolving the common technical contradiction in this field of "increased stability but decreased activity," thus ensuring its practical value.

[0051] According to an embodiment of this application, the method further includes: deleting amino acids 2 to 12 of wild-type streptavidin.

[0052] biomaterials In a third aspect of this application, a biological material is proposed, comprising any one of the following (i) to (iii): (i) a nucleic acid molecule that encodes the streptavidin mutant described in the first aspect; (ii) a vector carrying the nucleic acid molecule described in (i); (iii) Recombinant cells comprising the nucleic acid molecule described in (i) or the vector described in (ii), or expressing the streptavidin mutant described in the first aspect.

[0053] According to embodiments of this application, the above-mentioned biomaterial may further include at least one of the following technical features: According to embodiments of this application, the nucleic acid includes DNA or RNA.

[0054] It should be noted that, for the nucleic acids mentioned herein, those skilled in the art should understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases herein, the other complementary strand is also disclosed. Furthermore, the molecular sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0055] The vector according to the embodiments of this application carries the aforementioned nucleic acid molecule. When linking the nucleic acid molecule to the vector, the nucleic acid molecule can be directly or indirectly connected to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself, or they can be exogenous, i.e., not derived from the vector itself. Naturally, the nucleic acid molecule and the control elements only need to be operably connected.

[0056] In this article, "operably ligated" refers to ligating a foreign gene to a vector, enabling the control elements within the vector, such as transcriptional and translational control sequences, to perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids and bacteriophages. According to some specific embodiments of this application, after the vector is introduced into suitable recipient cells, the expression of the aforementioned streptavidin mutant can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of the streptavidin mutant.

[0057] According to embodiments of this application, the vector may refer to a cloning vector, which can be obtained by operatively ligating the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in this application is not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0058] In some optional embodiments of this application, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0059] In some optional embodiments of this application, the expression vector is a plasmid expression vector or a lentiviral expression vector.

[0060] The recombinant cells according to embodiments of this application carry the aforementioned nucleic acid molecules or the aforementioned vector; or, the recombinant cells express the streptavidin mutant described in the first aspect. Using this cell or host under suitable conditions, the aforementioned streptavidin mutant can be effectively expressed within the cell or host.

[0061] According to embodiments of this application, the cells are obtained by introducing the aforementioned vector into cells or a host.

[0062] It should be noted that the cells or hosts used in this application are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, and Trichoderma; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0063] In one optional embodiment of this application, the cells are mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, but do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0064] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the streptavidin mutant described in this application. Those skilled in the art will readily understand that suitable conditions for the expression of the streptavidin mutant include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the streptavidin mutant according to the specific environment of their laboratory.

[0065] Furthermore, those skilled in the art will understand that the features and advantages described above for the streptavidin mutant also apply to this biomaterial, and will not be repeated here.

[0066] complex In a fourth aspect of this application, a complex is proposed. According to an embodiment of this application, the complex comprises the streptavidin mutant described in the first aspect and a streptavidin conjugate; the streptavidin mutant and the streptavidin conjugate are linked together.

[0067] As previously known, the streptavidin mutant of this application possesses advantages such as high thermal stability and strong biotin-binding activity. Therefore, the complex containing the streptavidin mutant not only improves stability but also maintains or enhances biotin-binding activity, ensuring its practical value. It can be used for biological detection, molecular purification, nucleic acid analysis, drug delivery, and targeted drug therapy, and has been developed into molecular diagnostic tools, biosensors, or separation and purification materials with different temperature requirements.

[0068] According to embodiments of this application, the above-described complex may further include at least one of the following technical features: According to embodiments of this application, the streptavidin conjugate is selected from at least one of biotin or biotin-conjugated molecules, molecular probes, markers, and solid-phase supports.

[0069] In this article, the term "biotin," also known as vitamin B7 / vitamin H, refers to a small-molecule, water-soluble B vitamin with a molecular weight of only 244 Da, which has a non-covalent interaction with streptavidin. "Biotin-coupled molecules" refer to conjugated molecules formed by the covalent coupling of biotin to biomolecules such as proteins, nucleic acids, and antibodies, without significantly affecting the binding ability of the conjugated molecules to streptavidin.

[0070] In this paper, the term "molecular probe" refers to a complex formed by the molecular probe being coupled with a signal molecule or solid-phase carrier (e.g., by coupling the molecular probe with streptavidin, for example by modifying the probe with biotin to form a streptavidin) for the enrichment and detection of target molecules.

[0071] In this paper, the term "marker" refers to a class of substances that have properties that can be directly observed by the naked eye or detected or probing by instruments, such as luminescence, color development, radioactivity, etc., which enable qualitative or quantitative detection of the corresponding target.

[0072] In one optional embodiment of this application, the marker includes, but is not limited to, fluorescent markers, enzymes, etc.

[0073] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this application.

[0074] In one optional embodiment of this application, the fluorescent marker includes, but is not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3...). .5, Cy5, Cy5.5, Cy3, etc. or similar), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0075] In one optional embodiment of this application, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0076] In this application, the term "solid support" can refer to a substance that can be suspended or dispersed in a liquid phase (e.g., solid supports such as particles or magnetic beads), or a solid phase that can contain or carry a liquid phase (e.g., supports such as plates, membranes, or test tubes, as well as containers such as perforated plates, microfluidic paths, glass capillaries, nanopillars, or monolithic columns).

[0077] In one alternative embodiment of this application, the solid support is selected from microspheres, plates, and membranes.

[0078] In one optional embodiment of this application, the solid carrier includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0079] Furthermore, those skilled in the art will understand that the features and advantages described above for streptavidin mutants and biomaterials also apply to this complex, and will not be repeated here.

[0080] reagents or kits In a fifth aspect of this application, a reagent or kit is provided. According to embodiments of this application, the reagent or kit comprises the streptavidin mutant described in the first aspect, the biomaterial described in the third aspect, or the complex described in the fourth aspect.

[0081] As previously known, the streptavidin mutant of this application has advantages such as high thermostability and strong biotin-binding activity, which can improve stability while maintaining or enhancing biotin-binding activity. Therefore, reagents or kits containing the streptavidin mutant can be used for biological detection, molecular purification, nucleic acid analysis, drug delivery, and targeted drug therapy.

[0082] In this paper, kits or reagents do not need to have a box structure; they only need to be relatively independent and have suitable loading or containers, such as tubes, boxes, bottles, or cards. Some components are contained in different containers, and if permitted, some components may be combined in one container.

[0083] Furthermore, those skilled in the art will understand that the features and advantages described above for streptavidin mutants, biomaterials and complexes also apply to this reagent or kit, and will not be repeated here.

[0084] use In a sixth aspect of this application, the use of the streptavidin mutant described in the first aspect, the biomaterial described in the third aspect, the complex described in the fourth aspect, or the reagent or kit described in the fifth aspect in the preparation of products for biological detection, molecular purification, nucleic acid analysis, drug delivery, and targeted drug therapy is proposed.

[0085] According to embodiments of this application, the above-mentioned uses may further include the following technical features: In this paper, the term "biodetection" refers to the qualitative and quantitative detection of target molecules (such as proteins, nucleic acids, pathogens, and small molecule metabolites) in biological samples. For example, streptavidin mutants, by binding to biotinylated detection molecules (e.g., probes / antibodies), achieve multi-level signal amplification, thereby enabling the qualitative and quantitative detection of target molecules in biological samples. This can be used for disease diagnosis, food safety testing, environmental monitoring, and more.

[0086] According to embodiments of this application, the bioassay includes enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemistry, or flow cytometry. In all of these assays, the streptavidin mutant of this application can be used to directly or indirectly capture the target, thereby detecting the target.

[0087] In this paper, the term "molecular purification" refers to the isolation of target biomolecules (proteins, nucleic acids, etc.) from biological samples (e.g., cell lysates, blood, tissue homogenates). For example, streptavidin mutant-conjugated magnetic beads or chromatographic media can specifically capture biotinylated target molecules in samples and can be used for the affinity purification of biotinylated proteins and nucleic acids.

[0088] According to embodiments of this application, the molecular purification includes affinity chromatography, magnetic bead separation, and chip immobilization. Thus, the streptavidin mutant of this application can be used to directly or indirectly capture target biomolecules to obtain high-purity target biomolecules.

[0089] In this paper, the term "nucleic acid analysis" refers to the isolation of nucleic acids (DNA, RNA), which can be used for qualitative and quantitative analysis of nucleic acid molecules, gene sequencing, gene expression analysis, nucleic acid interaction studies, etc. For example, streptavidin magnetic bead mutants, by binding to biotinylated nucleic acid probes, can be used for mRNA capture in transcriptome sequencing, target gene enrichment in high-throughput sequencing, specific isolation of NAD+-capped RNA, and can also anchor biotinylated RNA for RNA-protein interaction analysis (such as RNA pull-down assays).

[0090] According to embodiments of this application, the nucleic acid analysis includes nucleic acid hybridization signal amplification, biological sequencing, and microarray chip detection. Thus, the streptavidin mutant of this application can be used to directly or indirectly capture nucleic acid molecules for analysis.

[0091] In this paper, the term "drug delivery" refers to the precise transport of drug molecules to the lesion site via carrier materials, controlling the drug release rate, reducing the toxic side effects of drugs on normal tissues, and can be used to treat diseases. For example, streptavidin magnetic bead mutants can serve as biotin-bridging media, enabling the coupling of biotinylated drugs (such as doxorubicin or siRNA) to the surface of nanocarriers (such as magnetic microspheres) to achieve drug carrier loading and complete targeted drug delivery.

[0092] In this article, the term "targeted therapy" refers to the specific accumulation of drugs at the lesion site (such as tumor tissue) guided by a targeting ligand, which precisely kills diseased cells and reduces damage to normal tissues, and can be used to treat diseases (such as cancer). For example, streptavidin-conjugated magnetic nanocarriers can bind to targeting ligands (such as folic acid, tumor-specific antibodies) and therapeutic drugs via biotin bridging, and actively accumulate in tumor tissue under the guidance of an external magnetic field, achieving active targeted therapy and significantly reducing systemic drug toxicity.

[0093] According to embodiments of this application, the products include, but are not limited to, reagents, kits, detection chips, and biosensors.

[0094] Furthermore, those skilled in the art will understand that the features and advantages described above for streptavidin mutants, biomaterials, complexes, and reagents or kits also apply to this use, and will not be repeated here.

[0095] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0096] Example 1: Design of a Streptavidin with High Thermal Stability Based on the structural analysis of wild-type streptavidin, amino acid site G48, located in the hydrophobic core, subunit interface, and key flexible loop region of the protein, was screened. Using site-directed mutagenesis, this amino acid was replaced with an amino acid that has higher hydrophobicity and is more likely to form hydrogen bonds or salt bridges, thus constructing several mutants: G48A (amino acid sequence as shown in SEQ ID NO:2), G48M (amino acid sequence as shown in SEQ ID NO:3), G48P (amino acid sequence as shown in SEQ ID NO:4), G48H (amino acid sequence as shown in SEQ ID NO:5), G48L (amino acid sequence as shown in SEQ ID NO:6), and G48D (amino acid sequence as shown in SEQ ID NO:9).

[0097] MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVDNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS (SEQ ID NO:9).

[0098] 1) Primer design: Open the pET-28a-SA-WT wild-type sequence file in SnapGene software and design primers at the G48 site. Extend 10-15 nucleotides to the left and right of this site, making each primer approximately 30 bp in length. No obvious dimer structure should form between the upstream and downstream primers. Purification was performed using PAGE. Specific primer sequences are shown in Table 1.

[0099] Table 1: Primer sequences

[0100] 2) PCR reaction system preparation. Prepare the PCR reaction system in a 0.2 mL PCR tube and mix all components thoroughly. The specific system preparation is shown in Table 2.

[0101] Table 2: PCR reaction system configuration

[0102] 3) Set the PCR instrument program to simulate tube temperature, with a volume of 50 μL, and start the 105℃ constant temperature hot cap mode. Specific PCR steps are shown in Table 3.

[0103] Table 3: PCR steps

[0104] 4) After the PCR reaction, add two volumes of anhydrous ethanol to the PCR sample, transfer the sample to a 1.5 mL microcentrifuge tube, mix well, and place in a -20°C freezer for 30 min. After the reaction, remove the sample. Centrifuge at 4°C and 12000 rpm for 10 min. After centrifugation, carefully aspirate the supernatant, add 10 μL of ddH2O, and repeatedly pipette to rinse the bottom and walls of the tube to dissolve the precipitate. Add 5 μL of the sample to DH5α competent cells, incubate on ice for 30 min, heat shock at 42°C for 45 s, incubate on ice for 2 min, add 500 μL of LB liquid medium, and incubate at 37°C and 220 rpm for 1 h to recover. Centrifuge the bacterial culture at 4000 rpm for 2 min. Discard the culture medium until approximately 50 μL remains. Spread the bacterial culture onto LB agar plates containing kanamycin resistance (final concentration 50 μg / mL) and incubate at 37°C. The next day, pick a single colony and transfer it to a shaker tube containing 5 mL of LB liquid medium. Incubate at 37°C for 8 hours at 220 rpm. Extract plasmids from the bacterial culture. Label the plasmids as pET-28a-SA-G48A and pET-28a-SA-G48M, respectively. Use universal sequencing primers T7 and T7ter for sequencing verification to ensure the correct sequence after the target site mutation.

[0105] Example 2: Preparation of streptavidin mutant 1. Taking the G48A mutant as an example, the recombinant pET-28a-SA-G48A-C6HIS (carrying 6 HIS tags at the C-terminus) verified by sequencing was transformed into BL21(DE3) competent cells and plated on LB agar plates with a final kanamycin resistance concentration of 50 μg / mL. The plates were then incubated overnight at 37°C. The next day, a single colony was picked and transferred to a 1L Erlenmeyer flask of LB agar with a final kanamycin resistance concentration of 50 μg / mL. The flask was placed in a shaker at 37°C and 220 rpm until the OD value reached 0.8-1.0. IPTG was added at a final concentration of 1 mM to induce expression. The induction temperature for pET-28a-SA-G48A-C6HIS (carrying 6 HIS tags at the C-terminus) was 42°C. After 4 hours of induction, the cells were harvested by centrifugation at 4000 r / min and stored at -20°C.

[0106] Resuspend 1 L of bacterial cells in approximately 50 mL of 1X PBS buffer (pH 7.2-7.4). After homogenizing using an IKA (T18digital) homogenizer, homogenize using an ATS (AH-NANO) at 800 bar for 5 min, until the sample is clear and non-viscous. Centrifuge at 12000 rpm for 30 min, discard the supernatant, and collect the precipitate.

[0107] Add 30 mL of 2M UREA buffer (50 mM Tris, 2 M UREA, pH 8.25), and resuspend using a homogenizer. Incubate at 37°C with shaking at 200 rpm for 20-30 min. Centrifuge at 12000 rpm for 30 min, discard the supernatant, and collect the precipitate.

[0108] Add 5-10 mL of 8M UREA buffer (50 mM PB, 8M UREA, pH 6.0), and resuspend using a homogenizer. Incubate at 37°C with shaking at 200 rpm for 1-2 hours. Centrifuge at 12000 rpm for 30 min and collect the supernatant. Detect the A280 absorbance of the G48A 8M urea-denatured sample using Nanodrop (Thermo Fisher Scientific), and calculate the molar and mass concentrations based on the extinction coefficient and molecular weight (see Table 4 below). Simultaneously, assess sample purity using SDS-PAGE electrophoresis. Add a certain volume of refolding buffer (50 mM Tris, 10 mM NaCl, pH 8.25) to a beaker to dilute the streptavidin mutant, ensuring a final protein concentration of 0.1-0.2 mg / mL in the refolding buffer to guarantee accurate folding and efficient refolding. Incubate overnight at 4°C with gentle stirring.

[0109] Table 4: Molecular weight and extinction coefficient of SA-G48A

[0110] Add 1-2 mL of Ni NTA affinity medium (purchased from Tiandi Renhe Company, catalog number SA00401L) to the above protein sample, mix well, and incubate for 2 hours at 4°C on a shaker. After binding, transfer the mixture to a 30 mL small gravity flow chromatography column and collect the flow-through (unbound contaminants) under natural gravity. Then, wash the bound proteins in the column with 1×PBS buffer (pH 7.4) to remove non-specific adsorbed impurities until the A280 absorbance of the eluent stabilizes. Prepare eluents: Using 2.5 M imidazole stock solution (pH 8.0) as the stock solution, dilute to prepare 1×PBS eluents (pH 7.2-7.4) containing 10 mM, 20 mM, 50 mM, and 300 mM imidazole, respectively. Add each gradient eluent to the column in ascending order of imidazole concentration, and collect the eluent fractions in segments. Nanodrop (ThermoFisher Scientific) was used to measure the A280 absorbance of each eluent component to determine that the target protein was enriched in the 300 mM imidazole eluent.

[0111] Finally, the buffer was replaced and preserved using a Hiload 16 / 600 Superdex 75 pg molecular sieve or a Desalting 16 / 10 desalting column pre-equilibrated with 1X PBS (pH 7.2-7.4). Protein was concentrated using a 10 kDa molecular weight cutoff concentration tube, and the A280 of the protein was determined using a Nanodrop (Thermo Fisher Scientific) microscope to calculate the protein concentration. The purity of the concentrated sample was assessed by SDS-PAGE electrophoresis.

[0112] 2. Based on step 2 of this embodiment, the mutant proteins (G48M, G48P, G48H, G48L, G48D) in Example 1 were prepared, and the buffer was replaced and preserved using a Hiload 16 / 600 Superdex 75 pg molecular sieve or a Desalting 16 / 10 desalting column pre-equilibrated with 1X PBS (pH 7.2-7.4). The protein was concentrated using a 10 kDa molecular weight cutoff concentration tube, and the A280 of the protein was determined by Nanodrop (Thermo Fisher Scientific) to calculate the protein concentration. The purity of the concentrated sample was detected by SDS-PAGE electrophoresis.

[0113] SDS-PAGE electrophoresis of SA-G48A mutant protein as follows: Figure 1 (From left to right: protein marker, unheated sample, and heated sample) As shown, the results of Hiload 16 / 600 superdex 75 pg molecular sieve tests for SA-G48A and SA-G48M mutants are as follows: Figure 2 and Figure 3 As shown in the figure. The results indicate that the purity of mutant proteins such as SA-G48A and SA-G48M is greater than 95%, excluding the influence of other contaminating proteins.

[0114] Example 3: Streptavidin mutant T m Measurement Based on the principle of fluorescence, the mechanism of action of specific fluorescent dyes (such as hydrophobic dyes like SYPRO Orange) is as follows: the fluorescence of dye molecules is weak (or quenched) in the free state; after the dye binds to hydrophobic residues on the surface of proteins (such as leucine, isoleucine, phenylalanine, etc.), the fluorescence signal is significantly enhanced. m During detection, as temperature increases, the protein's native conformation (folded state) dissociates into a denatured conformation (extended state), exposing more internal hydrophobic regions. This increases the amount of dye binding to the denatured protein, causing the fluorescence intensity to increase in an "S-shape" curve with increasing temperature. The midpoint temperature of the fluorescence signal change curve (i.e., the temperature at which the fluorescence intensity reaches 50% of its maximum change) is the protein's Tg. m (Midpoint temperature of denaturation).

[0115] The Thermo Fisher Scientific Protein Thermal Shift™ Dye Kit (catalog number: 4461146, hereinafter referred to as "PTS kit"), StepOnePlus real-time quantitative PCR instrument (instrument model: STEPONEPLUS), and StepOnePlus™ real-time quantitative PCR system were used to detect protein T. m The experimental steps are as follows: 1) Use an Axygen® 96-well flat-top polypropylene PCR microplate (catalog number: PCR-96-FLT-C, purchased from CorningLife Sciences). Taking the SA-G48A mutant as an example, prepare the reaction mixture according to the recommended proportions in the PTS kit instructions: 20 μL of reaction mixture per well, with a final protein concentration of 0.625 mg / mL, and three replicates per assay. Use wild-type SA as a control.

[0116] 2) On the StepOnePlus™ system, select the "Melt Curve" analysis module and set the "Continuous Temperature Sampling" mode. Temperature curve settings: 25°C for 2 minutes; start at 25°C and continuously increase to 95°C at a rate of 1% / min; hold at 95°C for 2 minutes. Start the test.

[0117] 3) After detection, the system automatically generates a fluorescence intensity-temperature curve (raw data) and a melting curve. The raw fluorescence curve is then subjected to first-derivative transformation (using a built-in software algorithm) to generate a "fluorescence intensity first derivative-temperature" curve. The temperature corresponding to the lowest value (inflection point) of the first-derivative curve is taken as the T temperature of the target protein. m Value (midpoint temperature of denaturation) for T values ​​of 3 parallel wells for each sample m The average value is taken as the T value of the protein. m value.

[0118] The temperature-fluorescence first derivative curve of some streptavidin mutants detected by the StepOnePlus™ system is shown in the figure below. Figure 4 and Figure 5 (The temperature corresponding to the lowest point of the temperature-fluorescence value first derivative curve in the figure is the T value of the protein.) m Value, T m A higher T value indicates a stronger resistance to heat denaturation and higher conformational stability of the protein; conversely, a lower T value indicates a lower protein stability. m As shown in the figure, the lower the value, the weaker the thermal stability. (SA mutant protein T) m A summary chart of the values ​​is shown below. Figure 6 (The pink color of the rectangular bars in the diagram represents T) mValues ​​superior to wild type; brown checkered histogram bars indicate T. m (The value is lower than that of wild type, and ND indicates that the protein cannot be purified and enriched, as shown in the figure.)

[0119] The results showed that, in addition to G48D, the T mutants of G48A, G48M, G48P, G48H, and G48L also showed similar results. m The values ​​were all superior to those of wild-type streptavidin.

[0120] Example 4: Detection of Biotin Binding Ability of Streptavidin Mutant HABA (2-hydroxyazobenzoic acid-4'-carboxylic acid) reagent provides a simple method for detecting the affinity of streptavidin for biotin over a wide range of pH and salt concentrations. HABA binds to streptavidin to form an orange-yellow colored complex with absorbance at 500 nm. Biotin competes with HABA, displacing HABA from the complex, resulting in a decrease in absorbance. A linear relationship exists between biotin concentration and absorbance, allowing the calculation of SA enzyme activity based on the amount of biotin used. Simultaneously, the IC50 (displacement rate of biotin to HABA in the HABA-SA complex) can be calculated. 50 By comparing the half-replacement rate (IC50) of SA wild-type and mutant strains, 50 The enzyme activity was measured to determine whether the mutant's biotin binding ability was the same as the wild type. The experimental steps are as follows: 1) Prepare a 10mM HABA solution: Weigh 24.2 mg of HABA, dissolve HABA in 1 mL of 0.25 M NaOH solution, and then dilute with 9 mL of 1X PBS (pH 7.2-7.4) to obtain a 10 mL 10mM HABA solution. Prepare a 10mM biotin solution: Weigh 24.4 mg of biotin, completely dissolve biotin in 1 mL of dimethyl sulfoxide (DMSO) solution, and then dilute with 9 mL of 1X PBS (pH 7.2-7.4) to obtain a 10 mL 10mM biotin solution. Then dilute with 1X PBS (pH 7.2-7.4) to obtain a 500 μM biotin titration solution for later use.

[0121] 2) The detection system configuration is shown in Table 5: a 100 μL reaction system is configured.

[0122] Table 5: Detection System

[0123] When performing biotin-to-HABA-SA replacement experiments, it is necessary to ensure that the number of SA protein molecules is consistent in each sample well. The results are shown in Table 6. Wild-type and SA-G48A mutant samples are used as examples. The background sample contains only HABA and buffer, without protein.

[0124] Table 6: Concentration, volume, and number of molecules in wild-type and mutant strains

[0125] The sample loading layout in the 96 wells is shown in Table 7: Table 7: Layout of 96 holes

[0126] 3) Open the microplate reader software and click "New". Set the microplate reader program, first select "96 WELLPLATE", change the detection "wavelength" to "500nm", click "A1,A12" in the upper right corner to select the measurement area, and finally click "Confirm".

[0127] 4) Add the prepared HABA-SA reaction solution to each well of a 96-well plate (100 μL). Let it stand for 2 minutes until the reaction solution returns to room temperature. Then, click "Confirm" and place the 96-well plate into a microplate reader to measure the absorbance at 500 nm. Record the initial value.

[0128] 5) Remove the 96-well plate and add 2 μL of 500 μM biotin solution to each well. Mix thoroughly with a pipette, check for air bubbles, and once stable, place the plate in the microplate reader. Press the start button and measure the absorbance at 500 nm. Record the data. Repeat the titration until the absorbance at 500 nm no longer decreases.

[0129] 6) Record the measured absorbance values ​​and the corresponding volumes of 500 μM biotin in a table. Divide the total μg of biotin when the absorbance no longer decreases by the mg of SA in 100 μL of the reaction system to obtain the activity unit of SA, U / mg.

[0130] OD_sample represents the detection value for each sample well. ΔOD_sample is the sample measurement value minus the HABA control group measurement value, which is the true value. OD_max is the maximum value when there is no biotin competitive substitution of HABA. ΔOD_max is OD_max minus the HABA control group measurement value. Record the ΔOD_sample for each sample well and the biotin concentration in the system for each titration.

[0131] The formula for calculating the replacement rate is: Biotin to HABA replacement rate = 100 [1 - (ΔOD_sample / ΔOD_max); Data was processed using GraphPad Prism software. Biotin concentration (μM) was plotted on the x-axis, and the corresponding biotin-HABA replacement rate on the y-axis. The x-axis data were then processed using log(x). The built-in function `Log(inhibitor) vs. Response -- Variable slope (four parameters)` was used, with constraints `Bottom=0` and `Top=100`. This yielded the half-maximum replacement rate (IC) of the samples. 50 (That is, the concentration corresponding to 50% HABA in HABA-SA replaced by biotin), the enzyme activity and replacement rate of wild-type and mutant can be compared. The enzyme activity results are shown in Table 8, and the replacement rate results are shown in Table 9. Figure 7 (The black dotted line graph represents the HABA replacement rate in the SA-HABA complex based on the biotin concentration of WT, and the rest represent the HABA replacement rate in the SA-HABA complex based on the biotin concentration of each mutant) and Table 9.

[0132] Table 8: Enzyme activities of wild-type streptavidin and mutants

[0133] Table 9: Half-replacement ratio of HABA in the biotin-HABA-SA complex

[0134] The results showed that the enzyme activities of both G48A and G48M mutants were higher than those of wild-type streptavidin to biotin.

[0135] The half-substitution rate of biotin for HABA in HABA-SA can indirectly indicate the affinity of the SA protein for biotin; the lower the half-substitution rate, the stronger the affinity of the SA protein for biotin. The results showed that the half-substitution rates of both the G48A and G48M mutants were lower than those of wild-type streptavidin.

[0136] Example 5: Mutant activity detection under heat treatment HABA (2-hydroxyazobenzo-4'-carboxylic acid) exhibits a characteristic absorption peak at 350 nm when in its free state. Using Ni-charged MagBeads carrying six HIS-tagged SA proteins, magnetic beads were added to a solution containing free HABA. The SA proteins on the magnetic beads bound and carried away the free HABA from the solution, resulting in magnetic separation. The change in the OD value at 350 nm after this process reflects the amount of HABA carried away by the SA proteins.

[0137] After adding a specific amount of biotin, due to its extremely high affinity for SA, biotin will competitively displace the HABA already bound to the magnetic beads. The free HABA in the solution increases again, and the OD value recovers. Calculating the amount of recovery at this point reflects the affinity of wild-type and mutant for biotin.

[0138] By pretreating magnetic beads coated with SA protein by heating (70℃, 10 min), the same operation was repeated to determine the biotin-to-HABA replacement ratio, and the stability of SA mutants and wild-types after heat treatment was compared.

[0139] Qualitative relationship: The displaced free HABA is reintroduced into the system, causing a recovery in the OD350 value. The magnitude of the change in OD value directly reflects the binding ability of SA protein and its mutants to ligands. The experimental procedure is as follows, taking SA-WT-C6HIS and SA-G48A-C6HIS as examples: 1) Prepare a 10mM HABA solution: Weigh 24.2 mg of HABA, dissolve HABA in 1 mL of 0.25 M NaOH solution, and then add 9 mL of 1X PBS (pH 7.2-7.4) to dilute to 10 mL of 10 mM HABA solution. Dilute further with 1X PBS to prepare a 300 μM HABA solution. 2) Prepare a 10mM biotin solution: Weigh 24.4 mg of biotin, completely dissolve biotin in 1 mL of dimethyl sulfoxide (DMSO) solution, and then add 9 mL of 1X PBS (pH 7.2-7.4) to dilute to 10 mL of 10 mM biotin solution. Dilute further with 1X PBS (pH 7.2-7.4) to prepare a 500 μM biotin titration solution.

[0140] 2) Pipette 100 μL of Ni-charged MagBeads suspension into two low-adsorption 1.5 mL microcentrifuge tubes (labeled WT group and G48A group, respectively). Place the tubes on a magnetic rack and allow the solution to clarify after magnetic separation. Aspirate the supernatant to preserve the solution. Add 1 mL of 1XPBS solution and rinse the magnetic beads by aspiration. After each rinse, place the tubes on a magnetic rack for magnetic separation for 2 min, and aspirate the supernatant. Repeat this rinsing step a total of 3 times. Add the measured amounts of protein solution and corresponding 1XPBS buffer to the tubes according to Table 10, ensuring that the total volume of solutions added is consistent.

[0141] Table 10: Coating protein content and total volume of wild-type and mutant strains

[0142] Add 600 μg of SA-WT-C6HIS protein to one tube of Ni magnetic beads and SA-G48A-C6HIS protein to another tube. Incubate in an inverted mixer at 4°C for 30 min. After incubation, magnetically separate the supernatant. Resuspend the magnetic beads in 100 μL of 1XPBS solution and place on ice. Zero the Nanodrop (Thermo Fisher Scientific) instrument using 1XPBS solution. Measure the absorbance of the collected supernatant at A280 and calculate the residual protein amount in the supernatant based on the extinction coefficient of the corresponding protein. Calculate the amount of magnetic beads bound by "total protein amount (600 μg) - residual protein amount in supernatant" to ensure complete and quantitative coating of the magnetic beads.

[0143] 3) Divide the coated WT magnetic beads into two tubes (300 μg protein per tube). Label one tube as WT-Heated and the other as WT-Non-Heated. Perform the same procedure for G48A magnetic beads. Place the heated group of magnetic beads in a 70°C thermostatic metal bath and heat for 10 min. Leave the non-heated group at room temperature. After the heating process is complete, centrifuge the entire solution in each tube until it reaches the bottom.

[0144] 4) Place the four magnetic beads on a magnetic rack. After complete magnetic separation, remove the supernatant. Add 100 μL of 300 μM HABA solution to each magnetic bead and mix carefully. Incubate at room temperature for 2 min. After incubation, place the four magnetic beads on a magnetic rack. After complete magnetic separation, add the samples to the 96-well plate in the order shown in Tables 11 and 12. The control is set as 100 μL of 300 μM HABA without magnetic bead treatment.

[0145] Table 11: Layout of 96 holes

[0146] The experimental setup for the G48M group was the same as that for the G48A group.

[0147] Table 12: Layout of 96 holes

[0148] 5) Open the ELISA reader software and click "New". Set the ELISA reader program: first select "96 WELLPLATE", change the detection "wavelength" to "350nm", set the reference "wavelength" to "620nm", click "A1,A5" in the upper right corner to select the measurement area, and finally click "Confirm". Record the initial value of the test sample OD_sample1 and the control sample OD_control.

[0149] 6) Remove the 96-well plate and aspirate the corresponding sample back into the magnetic beads, mixing thoroughly. Add 2 μL of 500 μM biotin solution to each magnetic bead tube and mix well. Incubate at room temperature for 2 min. After incubation, place the four magnetic bead tubes on a magnetic rack. Once magnetic separation is complete, add the samples back into the 96-well plate in sequence. For the control group (100 μL of 300 μM HABA, not treated with magnetic beads), add 2 μL of 500 μM biotin solution. After checking for air bubbles, place the plate in the microplate reader, press the start button, and measure the OD value at 350 nm. Record the data as OD_sample2. (OD value of control sample OD_control2).

[0150] 7) Calculate the biotin replacement rate. ΔOD_sample1 = OD_control - Initial value of sample OD_sample1. This indicates the amount of free HABA removed from the system by the SA protein on the Ni magnetic beads. It reflects the binding ability of mutants and wild-type to HABA. ΔOD_sample2 = OD_control2 - Initial value of sample OD_sample1 represents the amount of HABA still bound to the SA protein on the Ni magnetic beads in the system. OD_HABA replaced by biotin = ΔOD_sample1 - ΔOD_sample2 represents the amount of HABA returned to the solution system due to biotin competition. Biotin-HABA replacement rate = OD_HABA replaced by biotin / ΔOD_sample1. A higher biotin-HABA replacement rate indicates a better affinity of the SA protein for biotin.

[0151] The ΔOD values ​​of the G48A sample are shown in Table 13.

[0152] Table 13: ΔOD values ​​of G48A samples

[0153] The biotin-HABA replacement rate in the heat treatment experiment of G48A magnetic beads is shown in Table 14.

[0154] Table 14: Biotin-HABA replacement rate in G48A sample

[0155] The ΔOD values ​​of the G48M sample are shown in Table 15.

[0156] Table 15: ΔOD values ​​of G48M samples

[0157] The biotin-HABA replacement rate in the heat treatment experiment of G48M magnetic beads is shown in Table 16.

[0158] Table 16: Biotin-HABA replacement rate in G48M samples

[0159] The results showed that, under both heated and unheated conditions, the substitution rates of the G48A and G48M mutants were higher than those of wild-type streptavidin. Therefore, it can be concluded that the G48A and G48M mutants in this application exhibit superior affinity for HABA and biotin compared to wild-type streptavidin.

[0160] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0161] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A streptavidin mutant, characterized in that, Compared to wild-type streptavidin, the streptavidin mutant has a mutation, namely G48A / M / P / H / L, and the amino acid sequence of the wild-type streptavidin is shown in SEQ ID NO:

1.

2. The streptavidin mutant according to claim 1, characterized in that, Compared to wild-type streptavidin, the streptavidin mutant further lacks amino acids from position 2 to position 12.

3. The streptavidin mutant according to claim 1, characterized in that, The amino acid sequence of the streptavidin mutant is shown in any one of SEQ ID NO:2~6.

4. A method to improve the stability of streptavidin or T m The method of value, characterized in that, include: The 48th amino acid of wild-type streptavidin was mutated to enhance streptavidin T. m value; The mutation is G48A / M / P / H / L; The amino acid sequence of the wild-type streptavidin is shown in SEQ ID NO:

1.

5. The method according to claim 4, characterized in that, Further includes: It lacks amino acids 2 through 12 of wild-type streptavidin.

6. A biomaterial, characterized in that, The biomaterial includes any one of the following (i) to (iii): (i) A nucleic acid molecule that encodes the streptavidin mutant according to any one of claims 1 to 3; (ii) a vector carrying the nucleic acid molecule described in (i); (iii) Recombinant cells comprising the nucleic acid molecule described in (i) or the vector described in (ii), or expressing the streptavidin mutant described in any one of claims 1 to 3.

7. A complex, characterized in that, Includes the streptavidin mutant and streptavidin conjugate as described in any one of claims 1 to 3; The streptavidin mutant and streptavidin conjugate are linked.

8. The complex according to claim 7, characterized in that, The streptavidin conjugate is selected from at least one of biotin or biotin-conjugated molecules, molecular probes, markers, and solid-phase supports.

9. A reagent or kit, characterized in that, Includes the streptavidin mutant according to any one of claims 1 to 3, the biomaterial according to claim 6, or the complex according to any one of claims 7 to 8.

10. Use of the streptavidin mutant of any one of claims 1 to 3, the biomaterial of claim 6, the complex of any one of claims 7 to 8, or the reagent or kit of claim 9 in the preparation of products for biological detection, molecular purification, nucleic acid analysis, drug delivery, and targeted drug therapy.

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