A fusion protein for surface quantitative modification of single molecule force spectroscopy and application thereof

Abstract

The present application relates to the technical field of single molecule biophysics and biochemistry, and particularly relates to a fusion protein for surface quantitative modification of single molecule force spectrum and application. The fusion protein comprises a specific binding domain Catcher and / or a short peptide tag Tag, and further comprises an enhanced green fluorescent protein eGFP fusion domain. The present application introduces an enhanced green fluorescent protein (eGFP) fusion domain in a single molecule force spectrum system, and the Catcher and / or Tag are fused with the eGFP. The Catcher fused with the eGFP can realize direct quantitative surface fixation density through fluorescence. The Tag fused with the eGFP evaluates the connection activity of the surface protein. After the fusion protein is fixed on an aminated substrate, the effective connection point number in unit area can be characterized in situ and quantitatively through a fluorescence microscope, and the biological activity can be evaluated, so as to realize accurate control and prediction of the single molecule force spectrum experimental conditions, and greatly improve the experimental success rate and repeatability.

Description

Technical Field

[0001] This invention relates to the fields of single-molecule biophysics and biochemistry, specifically to a fusion protein for quantitative surface modification using single-molecule force spectroscopy and its applications. Background Technology

[0002] Single-molecule force spectroscopy is a core research tool for characterizing the intrinsic properties and dynamic behavior of biomolecules. Representative techniques include magnetic tweezers, optical tweezers, and atomic force microscopy (AFM). This type of technology can apply precise and controllable external forces to biomolecules such as proteins and nucleic acids at the single-molecule level, capturing their mechanical response signals in real time. This allows for the analysis of core information such as the mechanical properties, folding / unfolding dynamics, intermolecular interactions, and conformational dynamics of biomolecules, providing irreplaceable application value in basic life science research, drug development, and biomedical detection.

[0003] The core prerequisite and key step in single-molecule force spectroscopy is the specific anchoring of the target biomolecule in single-molecule form: one end of the target molecule is stably fixed to the surface of a solid substrate, while the other end is specifically connected to the force sensing unit of the force spectroscopy device (such as an AFM tip, microspheres captured by optical tweezers, or magnetic beads manipulated by magnetic tweezers). The performance of the anchoring system directly determines the effectiveness, data reliability, and reproducibility of the experiment.

[0004] Peptide-protein specific covalent coupling systems (commonly known in the industry as Cater-Tag systems) are a class of genetically engineered biological tools that enable site-specific irreversible covalent coupling. These systems are derived from the Ig-like domains of pili proteins or surface adhesion proteins in Gram-positive bacteria. Through rational artificial splitting and directed evolution, they are transformed into two completely complementary functional fragments: one is a short peptide tag (Tag) containing only 5-15 amino acids, with an extremely small molecular weight. This tag can be precisely modified into the N-terminus, C-terminus, or internal flexible loop region of the target protein through gene fusion, with almost no impact on the folding and physiological function of the target protein; the other is a specific binding domain (Catcher) with a molecular weight of approximately 10-15 kDa. This domain can spontaneously react with the homologous Tag under physiologically mild conditions, forming an irreversible intermolecular heteropeptide bond between the Cater and the Tag without the need for exogenous catalysts, activators, or strict reaction environment control. Currently, several mature and fully orthogonal pairing schemes have been developed for this type of system, with representative systems including SpyCatcher / SpyTag, DogCatcher / DogTag, SnoopCatcher / SnoopTag, SdyCatcher / SdyTag, and Pilin-C / Isopeptag. These systems possess core advantages such as tolerable reaction conditions, high coupling conversion rates, and strong site controllability, perfectly matching the anchoring requirements of single-molecule force spectroscopy experiments, and are therefore widely used in such experiments. Its general application method is as follows: the Tag is modified to a specific site on the target biomolecule through gene fusion, and the corresponding Cater domain is modified to the surface of a solid substrate (glass, silica, mica, gold sheet, etc.) for single-molecule force spectroscopy through chemical cross-linking. Through the specific covalent reaction between the two, the target molecule is stabilized on the substrate surface and fixed at a specific site. At the same time, taking advantage of the complete orthogonality between different Cater-Tag systems, different Tag tags can be fused to both ends of the target molecule, and the corresponding Cater domains can be modified on the substrate and the force sensing unit, respectively, to achieve bi-terminal orthogonal covalent anchoring of the target molecule, further avoiding non-specific binding and multi-molecule interference.

[0005] However, the inventors of this application discovered in their research that existing technologies, when applying various Cater-Tag systems to single-molecule force spectroscopy experiments, suffer from insurmountable core technical bottlenecks, severely restricting the controllability, success rate, and reproducibility of the experiments. First, the immobilization efficiency and activity retention rate of Cater domains on commonly used solid substrates for single-molecule force spectroscopy, such as glass, silica, and mica, fluctuate greatly due to the coupling influence of various factors, including surface chemical treatment processes, crosslinking agent type and parameters, and reaction environment. Second, existing technologies lack in-situ, quantitative characterization methods for the effective active sites of Cater domains on the substrate surface. Existing conventional characterization methods, including X-ray photoelectron spectroscopy (XPS), ellipsometry, BCA protein quantification, and fluorescently labeled total protein imaging, can only detect the total amount of Cater protein coupled to the substrate surface and cannot distinguish between effective binding sites and inactive, denatured, or misoriented proteins. The core influencing factor in single-molecule force spectroscopy experiments is precisely the areal density of the effective active binding sites on the substrate surface, rather than the total amount of coupled protein. Meanwhile, existing characterization methods are mostly in vitro and non-in-situ detection methods. The detection environment differs significantly from the in-situ liquid-phase environment of single-molecule force spectroscopy experiments. Therefore, the characterization results cannot accurately reflect the effective site conditions under experimental conditions and cannot provide quantitative basis for optimizing experimental conditions. These shortcomings directly lead to uncontrollable density of active effective connection points of the Catcher domain on the substrate surface in existing technologies. Experimenters can only rely on experience to repeatedly adjust surface modification conditions through trial and error, making it impossible to establish a standardized and quantitative sample preparation process. This results in extremely poor batch-to-batch consistency in single-molecule force spectroscopy experiments, and the success rate and repeatability cannot be effectively guaranteed.

[0006] Therefore, developing a method that can quantitatively characterize the surface catcher fixation density and its linkage activity in situ is crucial for promoting the standardization and reliable application of single-molecule force spectroscopy. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a fusion protein for quantitative surface modification by single-molecule force spectroscopy and its application.

[0008] In a first aspect, the present invention provides a fusion protein for single-molecule force spectroscopy-based quantitative surface modification, the fusion protein comprising a specific binding domain Catcher and / or a short peptide tag Tag, and further comprising an enhanced green fluorescent protein (eGFP) fusion domain.

[0009] Preferably, the fusion protein is a fusion of the specific binding domain Catcher and the enhanced green fluorescent protein eGFP fusion domain.

[0010] Preferably, the specific binding domain Cater is a SpyCatcher domain, and the C-terminus of the SpyCatcher domain is fused with eGFP.

[0011] Preferably, the fusion protein is a fusion of a short peptide tag (Tag) and an enhanced green fluorescent protein (eGFP) fusion domain.

[0012] Preferably, the short peptide tag is SpyTag, and the SpyTag fused with the enhanced green fluorescent protein eGFP fusion domain can be specifically covalently linked to SpyCatcher.

[0013] In a second aspect, the present invention provides an application of using the fusion protein described above for single-molecule force spectroscopy experiments.

[0014] A third aspect of the present invention provides a method for surface immobilization and quantitative density characterization in single-molecule force spectroscopy experiments, comprising the following steps: immobilizing the fusion protein as described above on a substrate surface, and quantitatively characterizing the surface modification density by detecting the eGFP fluorescence intensity.

[0015] A fourth aspect of the present invention provides a method for evaluating the linkage activity of single-molecule force spectroscopy experiments, comprising the following steps: (1) The fusion protein formed by fusing the short peptide tag Tag with the enhanced green fluorescent protein eGFP fusion domain was fixed on the substrate surface and stored under cold storage for a certain period of time; (2) Attach fluorescent labeling units to the Catcher domain protein and incubate them with the substrate of the immobilized fusion protein from step (1); (3) After incubation, the stability of its linkage activity was evaluated by fluorescence colocalization analysis; The fluorescent labeling unit is a fluorescent functional unit of enhanced green fluorescent protein (EGFP) that can be stably linked to the Catcher domain protein through covalent coupling or gene fusion, and whose excitation and emission spectra are fixed to the solid substrate surface without spectral crosstalk. The short peptide tag in step (1) and the Catcher domain protein in step (2) are a completely complementary pairing system.

[0016] Preferably, the fluorescent labeling unit is a near-infrared organic fluorescent dye.

[0017] Preferably, in step (3), the green channel and the red channel are acquired simultaneously by TIRF microscopy, and the percentage of SpyCatcher molecules that still have binding activity on the surface is calculated by analyzing the co-localization of the fluorescence signals of the two channels.

[0018] This invention introduces an enhanced green fluorescent protein (eGFP) fusion domain into a single-molecule force spectroscopy system. By fusing the Cater and / or Tag with eGFP, the Cater-eGFP fusion allows for direct quantitative measurement of surface immobilization density via fluorescence, while the Tag-eGFP fusion assesses the binding activity of surface proteins. After immobilizing this fusion protein on an aminated substrate, the number of effective binding sites per unit area can be characterized in situ and quantitatively using fluorescence microscopy, and its biological activity can be evaluated. This enables precise control and prediction of single-molecule force spectroscopy experimental conditions, significantly improving experimental success rate and reproducibility.

[0019] Specifically, the beneficial effects of the present invention include: 1. Precise quantification: For the first time, in-situ and absolute quantification of active bonding sites on the surface of single-molecule force spectrum has been achieved, transforming experimental conditions from empirical exploration to precise and controllable conditions.

[0020] 2. Activity monitoring: It can assess the long-term stability and functional activity of surface-fixed proteins and provide early warning of experimental failures caused by protein inactivation.

[0021] 3. Non-invasive: The fusion of eGFP does not interfere with the core covalent linking function of SpyCatcher / SpyTag (see specific implementation verification), ensuring that the reporting system does not affect the target biological process.

[0022] High versatility: Applicable to all single-molecule technology platforms based on Catcher / Tag specific binding systems and similar chemical immobilization, such as magnetic tweezers, optical tweezers, atomic force microscopy, etc. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0024] Figure 1 These are denaturing and non-denaturing gel electrophoresis images used in embodiments of the present invention to verify the function of the fusion protein. In the image: non-reducing SDS-PAGE. M: Protein Marker; Lane 1: SpyTag-eGFP (30.4 kDa); Lane 2: SpyCatcher-C-eGFP (47.3 kDa); Lane 3: Mixture of lanes 1 and 2 after incubation, showing a covalently linked product band at ~77.7 kDa.

[0025] Figure 2This is a schematic diagram illustrating the immobilization and function of the SpyCatcher-eGFP and eGFP-SpyTag fusion proteins on an amino-coated glass surface, in a preferred embodiment of the present invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0027] A fusion protein for single-molecule force spectroscopy-based quantitative surface modification, the fusion protein comprising a specific binding domain Catcher and / or a short peptide tag Tag, and further comprising an enhanced green fluorescent protein (eGFP) fusion domain.

[0028] In this system, the specific binding domain Cater and the short peptide tag are two separate parts. When they meet, they spontaneously form a stable isopeptide bond. Several mature and fully orthogonal pairing schemes have been developed for this type of system, with representative systems including SpyCatcher / SpyTag, DogCatcher / DogTag, SnoopCatcher / SnoopTag, SdyCatcher / SdyTag, and Pilin-C / Isopeptag, which are widely used in single-molecule force spectroscopy experiments. The specific binding domain Cater and / or short peptide tag selected in the fusion protein of this application are the Cater and / or Tag in the Cater-Tag system used for single-molecule force spectroscopy experiments, for the characterization of quantitative surface modifications in the corresponding single-molecule force spectroscopy experiments.

[0029] Among them, enhanced green fluorescent protein (eGFP) is a fluorescent reporter protein. In this application, eGFP provides a direct fluorescence signal for real-time monitoring of modification efficiency and quantitative analysis of surface density.

[0030] In some embodiments of this application, the fusion protein is a fusion of the specific binding domain Catcher and the enhanced green fluorescent protein eGFP fusion domain.

[0031] In some embodiments of this application, the specific binding domain Cater is a SpyCatcher domain, and the C-terminus of the SpyCatcher domain is fused with eGFP.

[0032] In some embodiments of this application, the fusion protein is a fusion of a short peptide tag (Tag) and an enhanced green fluorescent protein (eGFP) fusion domain.

[0033] In some embodiments of this application, the short peptide tag is SpyTag, and the SpyTag fused with the enhanced green fluorescent protein eGFP fusion domain can be specifically covalently linked to SpyCatcher.

[0034] Application of single-molecule force spectroscopy experiments using the fusion protein described above.

[0035] In some embodiments of this application, a method for surface immobilization and density quantitative characterization of single-molecule force spectroscopy experiments is further provided, which includes the following steps: immobilizing the fusion protein as described above on the substrate surface, and quantitatively characterizing the surface modification density by detecting the eGFP fluorescence intensity.

[0036] In some embodiments of this application, a method for evaluating the linkage activity of single-molecule force spectroscopy experiments is further provided, including the following steps: (1) The fusion protein formed by fusing the short peptide tag Tag with the enhanced green fluorescent protein eGFP fusion domain was fixed on the substrate surface and stored under cold storage for a certain period of time; (2) Attach fluorescent labeling units to the Catcher domain protein and incubate them with the substrate of the immobilized fusion protein from step (1); (3) After incubation, the stability of its linkage activity was evaluated by fluorescence colocalization analysis.

[0037] The short peptide tag in step (1) and the Catcher domain protein in step (2) are a completely complementary pairing system.

[0038] Specifically, refrigerated storage for a certain period of time is used to simulate the regular usage cycle of real experimental scenarios. The preparation process of glass slides for single-molecule force spectroscopy experiments is complex and has a high risk of batch fluctuations. Laboratories generally adopt the "batch preparation, refrigerated storage, and on-demand use" model. Specifically, the storage time can be set to 7 days, 14 days, or 30 days according to the regular experimental requirements to cover the needs from short-term validation to long-term stability assessment. The refrigeration temperature is strictly controlled at 2–8℃, which meets the non-destructive refrigeration conditions commonly used for biological samples.

[0039] Specifically, the fluorescent labeling unit is a fluorescent functional unit that can be stably linked to the Catcher domain protein through covalent coupling or gene fusion, and whose excitation and emission spectra are fixed to the solid substrate surface without spectral crosstalk of enhanced green fluorescent protein (EGFP). It can be a near-infrared dye, such as Alexa Fluor or Cy series near-infrared dyes, or other fluorescent probes that can exhibit red fluorescence signals, such as gene-encoded red fluorescent protein fusion labels or near-infrared quantum dot (QD) labels.

[0040] In some embodiments of this application, the fluorescent labeling unit is a near-infrared organic fluorescent dye. Specifically, one embodiment of this application uses the red fluorescent dye Alexa Fluor 647.

[0041] In some embodiments of this application, in step (3), the green channel and the red channel are acquired simultaneously by a TIRF microscope, and the percentage of SpyCatcher molecules that still have binding activity on the surface is calculated by analyzing the co-localization of the fluorescence signals of the two channels.

[0042] The following are some specific embodiments of this application: Example 1: Expression, purification and functional verification of fusion protein Plasmids encoding SpyCatcher-eGFP and eGFP-SpyTag were constructed using molecular cloning technology and induced for expression in *E. coli*. The target fusion proteins were purified using nickel column affinity chromatography and molecular sieve chromatography. The specific procedures followed standard operating protocols for molecular cloning, induced expression, and protein purification.

[0043] To verify whether eGFP fusion affects SpyCatcher function, the following binding experiment was performed: Purified SpyTag-eGFP protein and SpyCatcher-C-eGFP protein were mixed in equimolar amounts and incubated at room temperature for 1 hour, followed by non-reducing SDS-PAGE analysis. The results are as follows: Figure 1 As shown, a significant new band appeared at approximately 77.7 kDa in the sample lanes after incubation (corresponding to the covalent complex of SpyTag-eGFP and SpyCatcher-C-eGFP), while the individual SpyTag-eGFP (30.4 kDa) and SpyCatcher-C-eGFP (47.3 kDa) bands weakened. This result conclusively demonstrates that the fusion of eGFP into the C-terminus of SpyCatcher does not interfere with its efficient and specific covalent linkage with SpyTag.

[0044] Example 2: Surface fixation and quantitative density characterization Glass coverslips were cleaned with oxygen plasma and then immersed in an ethanol solution of APTES to achieve surface amination. The amination slides were reacted with the cross-linking agent SMCC, and then incubated with the SpyCatcher-C-eGFP fusion protein, allowing the protein to be covalently fixed to the slide surface via its N-terminus. After washing to remove unbound protein, the slides were placed under a total internal reflection fluorescence microscope. Figure 2As shown in the schematic diagram, each fusion protein on the surface emits green fluorescence. By capturing multiple fields of view and calibrating using known single-molecule eGFP fluorescence intensity, image analysis software can automatically identify and count the number of fluorescent spots per unit area, thereby directly calculating the surface fixation density of SpyCatcher (e.g., molecules per square micrometer). By adjusting the protein incubation concentration or time, the density can be precisely controlled within an ideal range (e.g., 0.1–1 molecules / μm²), providing an optimal surface for single-molecule experiments.

[0045] Example 3: Ligation activity assessment (for SpyCatcher-C-eGFP-SpyTag) After storing glass slides immobilized with eGFP-SpyTag (prepared according to the method in Example 2) at 4°C for one week, their ligation activity was evaluated. The slides were incubated with a solution of soluble SpyCatcher protein labeled with the red fluorescent dye Alexa Fluor 647. Because the C-terminus of the fusion protein on the slide surface carries a SpyTag, it binds to the labeled SpyCatcher in the solution. After washing, both the green channel (eGFP, labeling all immobilized sites) and the red channel (Alexa Fluor 647, labeling active sites) were simultaneously acquired using a TIRF microscope. Analysis of the co-localization of the fluorescence signals in the two channels allowed for the calculation of the percentage of SpyCatcher molecules still possessing ligation activity on the surface. This percentage of activity serves as a key indicator for predicting the subsequent ligation efficiency with the target protein.

[0046] Comparative Example: Uncertainty of Traditional Methods Wild-type SpyCatchers without fused eGFP were immobilized on a glass slide surface following the same procedure as in Example 2. Since direct observation is not possible, the actual immobilization density on the surface cannot be determined. In single-molecule magnetic tweezers experiments, a significant amount of time is typically spent repeatedly testing different fields of view to find a "suitable" magnetic sphere-surface connection, resulting in low success rates and poor reproducibility. However, using the method of this invention, the suitability of the surface density can be confirmed before the experiment, thus avoiding blind experimentation.

[0047] It will be understood by those skilled in the art that the above descriptions are merely preferred examples of the invention and are not intended to limit the invention. Although the invention has been described in detail with reference to the foregoing examples, those skilled in the art can still modify the technical solutions described in the foregoing examples or make equivalent substitutions for some of the technical features. All modifications and equivalent substitutions made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A fusion protein for quantitative surface modification using single-molecule force spectroscopy, characterized in that, The fusion protein includes a specific binding domain Catcher and / or a short peptide tag Tag, as well as an enhanced green fluorescent protein (eGFP) fusion domain.

2. The fusion protein according to claim 1, characterized in that, The fusion protein is a fusion of the specific binding domain Catcher and the enhanced green fluorescent protein eGFP fusion domain.

3. The fusion protein according to claim 2, characterized in that, The specific binding domain Catcher is the SpyCatcher domain, and the C-terminus of the SpyCatcher domain is fused with eGFP.

4. The fusion protein according to claim 1, characterized in that, The fusion protein is a short peptide tag (Tag) fused with an enhanced green fluorescent protein (eGFP) fusion domain.

5. The fusion protein according to claim 4, characterized in that, The short peptide tag is SpyTag, which is fused with the enhanced green fluorescent protein (eGFP) fusion domain and can be specifically covalently linked to SpyCatcher.

6. Application of the fusion protein as described in any one of claims 1-5 for single-molecule force spectroscopy experiments.

7. A method for surface immobilization and quantitative density characterization in single-molecule force spectroscopy experiments, comprising the following steps: The fusion protein as described in claim 2 or 3 is immobilized on the substrate surface, and the surface modification density is quantitatively characterized by detecting the eGFP fluorescence intensity.

8. A method for evaluating the linkage activity in single-molecule force spectroscopy experiments, characterized in that, Includes the following steps: (1) The fusion protein formed by fusing the short peptide tag Tag with the enhanced green fluorescent protein eGFP fusion domain was fixed on the substrate surface and stored under cold storage for a certain period of time; (2) Attach fluorescent labeling units to the Catcher domain protein and incubate them with the substrate of the immobilized fusion protein from step (1); (3) After incubation, the stability of its linkage activity was evaluated by fluorescence colocalization analysis; The fluorescent labeling unit is a fluorescent functional unit of enhanced green fluorescent protein (EGFP) that can be stably linked to the Catcher domain protein through covalent coupling or gene fusion, and whose excitation and emission spectra are fixed to the solid substrate surface without spectral crosstalk. The short peptide tag in step (1) and the Catcher domain protein in step (2) are a completely complementary pairing system.

9. The method for evaluating the linkage activity of single-molecule force spectroscopy experiments according to claim 8, characterized in that, The fluorescent labeling unit is a near-infrared organic fluorescent dye.

10. The method for evaluating the linkage activity of single-molecule force spectroscopy experiments according to claim 9, characterized in that, In step (3), the green and red channels are acquired simultaneously using a TIRF microscope. By analyzing the co-localization of the fluorescence signals in the two channels, the percentage of SpyCatcher molecules that still have binding activity on the surface is calculated.

Images