Target protein labeling or tracing composition and method

Abstract

The present invention relates to the field of protein labeling or tracing, and particularly to a composition and method for labeling or tracing a target protein. The method first analyzes the structure of the target protein and identifies a loop sequence on the protein surface that faces outward. Then, through molecular cloning methods, a small tag (such as an HA tag) with flexible amino acid linker peptides at both ends is inserted into the middle of the loop sequence. Finally, a nanobody fused to the small tag and fused to a fluorescent protein is expressed. This method utilizes the nanobody's ability to specifically recognize the target protein surface tag to achieve intracellular tracing of the target protein.

Description

Technical Field

[0001] The present invention relates to the field of protein labeling or tracing, and in particular to a labeling or tracing composition and method for a target protein. Background Art

[0002] In order to analyze the dynamic localization of proteins in cells, the target protein needs to be fluorescently labeled and imaged and observed using a live-cell fluorescence microscope. Traditional fluorescent labeling methods are often achieved by adding fluorescently labeled proteins to the C-terminus and N-terminus of the target protein. However, adding fluorescently labeled proteins to the C-terminus or N-terminus of the protein may affect the stability and function of the target protein. For example: the C-terminus or N-terminus of the target protein may be inside the protein structure, and the connection of fluorescently labeled proteins will destroy the protein structure; the C-terminus or N-terminus of the target protein may also interact with other proteins, and the connection of fluorescently labeled proteins will destroy protein interactions; the C-terminus or N-terminus of the target protein may mediate direct interactions with cellular substructures or organelles, and the connection of fluorescently labeled proteins will affect the accurate localization of the target.

[0003] Traditional fluorescent labeling methods involve directly attaching a fluorescent marker protein to the C- or N-terminus of the target protein, but this approach is not suitable for all proteins. Existing approaches involve directly inserting the fluorescent marker protein into the target protein. However, direct insertion of the fluorescent marker protein into the target protein often compromises its function. Because fluorescent marker proteins are often globular proteins consisting of approximately 200 amino acids, inserting this larger fluorescent marker protein can affect the structural stability of the target protein, potentially leading to loss of function.

[0004] In summary, using traditional methods to fluorescently label target proteins can disrupt their structure and localization within cells, thereby affecting their function and even normal cell proliferation. Therefore, providing a new method for fluorescently labeling protein surfaces is of great practical significance. Summary of the Invention

[0005] In view of this, the present invention does not label the target protein with fluorescent protein at the C-terminus or N-terminus, but instead connects the label protein to the protein surface (in the middle of the protein sequence) by referring to the protein structure, thereby achieving fluorescent labeling of the target protein.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides the use of a small tag with flexible amino acid connecting peptide segments at both ends and a small tag nanobody fused with a fluorescent protein in target protein labeling or tracing.

[0008] In some specific embodiments of the present invention, the small tag with flexible amino acid connecting peptide segments at both ends includes an HA tag; the small tag nanobody fused with a fluorescent protein includes an HA nanobody.

[0009] In some specific embodiments of the present invention, the insertion position of the small tag with flexible amino acid connecting peptide segments at both ends includes any position of the loop sequence facing outward on the surface of the target protein.

[0010] In some specific embodiments of the present invention, the target protein includes one or more of Dnm1, Mal3, and Nda3;

[0011] When the target protein is Dnm1, the insertion position includes between T680 and M681; or

[0012] When the target protein is Mal3, the insertion position includes between T222 and S223; or

[0013] When the target protein is Nda3, the insertion position includes between Y36 and H37.

[0014] In a second aspect, the present invention also provides a target protein labeling or tracing composition, comprising a small tag with flexible amino acid connecting peptide segments at both ends and a small tag nanobody fused with a fluorescent protein.

[0015] In some specific embodiments of the present invention, the small tag with flexible amino acid connecting peptide segments at both ends includes an HA tag; the small tag nanobody fused with a fluorescent protein includes an HA nanobody.

[0016] In some specific embodiments of the present invention, the insertion position of the small tag with flexible amino acid connecting peptide segments at both ends includes any position of the loop sequence facing outward on the surface of the target protein.

[0017] In some specific embodiments of the present invention, the target protein includes one or more of Dnm1, Mal3, and Nda3;

[0018] When the target protein is Dnm1, the insertion position includes between T680 and M681; or

[0019] When the target protein is Mal3, the insertion position includes between T222 and S223; or

[0020] When the target protein is Nda3, the insertion position includes between Y36 and H37.

[0021] In a third aspect, the present invention further provides use of the labeling or tracing composition in preparing a kit or device for labeling or tracing a target protein.

[0022] In a fourth aspect, the present invention further provides a kit or device comprising the marking or tracing composition and an acceptable carrier or auxiliary material.

[0023] In a fifth aspect, the present invention further provides a target protein labeling or tracing method based on the labeling or tracing composition or the kit, comprising the following steps:

[0024] Step 1: Obtaining the outward-facing loop sequence of the target protein;

[0025] Step 2: inserting the small tag with flexible amino acid connecting peptide segments at both ends into the site of the loop sequence;

[0026] Step 3: Use the small tag nanobody of the fusion fluorescent protein corresponding to the small tag with flexible amino acid connecting peptide segments at both ends to identify, locate and track the target protein.

[0027] In some specific embodiments of the present invention, the target protein includes one or more of Dnm1, Mal3, and Nda3;

[0028] When the target protein is Dnm1, the insertion position includes between T680 and M681; or

[0029] When the target protein is Mal3, the insertion position includes between T222 and S223; or

[0030] When the target protein is Nda3, the insertion position includes between Y36 and H37.

[0031] To overcome the challenge of labeling the target protein at its C- or N-terminus, the present invention provides a novel target protein labeling method. First, the target protein's structure is analyzed to identify a loop sequence oriented outward on the protein's surface. Then, through molecular cloning, a small tag (such as an HA tag) with flexible amino acid linker peptides at both ends is inserted into the center of this loop sequence. Finally, a small tag-containing nanobody (such as an HA nanobody) fused to a fluorescent protein is expressed. This method leverages the nanobody's ability to specifically recognize the target protein's surface tag, enabling intracellular tracking of the target protein.

[0032] The beneficial effects of the present invention include but are not limited to:

[0033] (1) The N- and C-termini of proteins such as Dnm1, Mal3, and Nda3 cannot be labeled, as labeling would affect protein function. To address this problem, we developed a novel method described in this invention that allows for labeling of these proteins without affecting their function.

[0034] (2) The special design is the key point of the present invention. We refer to the protein structure information to find the rings that are fully exposed on the surface of the protein, and perform hydrophobicity and hydrophilicity analysis on the amino acids on the rings. Then, we use CRISPR-Cas9 gene editing technology to insert small tags recognized by nanobodies at the sites in the protein surface rings, and then use the corresponding fluorescent protein fused nanobody (such as nanobody-GFP) to identify and locate the target protein that is surface-labeled with the nanobody recognizing the small tag (see Figure 1 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0036] Figure 1 The experimental process of the present invention is shown; wherein, (1) CRISPR technology is used to connect a small tag of a nanobody with flexible amino acid connecting peptides at both ends to a loop sequence on the surface of an endogenous target protein facing outward; (2) the nanobody sequence is connected to a vector containing green fluorescent protein (GFP) to obtain a plasmid of nanobody-green fluorescent protein (abbreviated as NB-GFP); (3) the NB-GFP plasmid is transformed or transfected into a cell in which a small tag of a nanobody is connected to a small tag facing outward on the surface of an endogenous target protein, and the plasmid stably expresses NB-GFP; (4) finally, NB-GFP specifically recognizes the small tag of the nanobody, thereby achieving tracing of the target protein;

[0037] Figure 2 This indicates that the C-terminal or N-terminal tagging of Dnm1 affects its function; Figure 2In A: The top row of images shows wild-type (WT) yeast (left) and dnm1 gene knockout (dnm1△) ​​yeast (right). Mitotracker is a mitochondrial dye that can dye mitochondria red. In wild-type yeast, mitochondria are tubular networks with obvious breaks (pointed by green arrows); in dnm1 gene-deficient yeast, mitochondria show no signs of fission and become a single tubular network; the middle and bottom rows show images of Dnm1 labeled at both ends; the middle row shows images of Dnm1 labeled with GFP at both ends. In yeast with Dnm1 C-terminus labeled with GFP (left), Dnm1-GFP is localized to mitochondria, but the mitochondrial morphology is abnormal, and there are no signs of mitochondrial fission; in yeast with Dnm1 N-terminus labeled with GFP (right), GFP-Dnm1 is localized to mitochondria, but the mitochondrial morphology is abnormal, and there are no signs of mitochondrial fission; the bottom row shows images of Dnm1 labeled with HA at both ends. In yeast with Dnm1 C-terminus labeled with HA (left), the mitochondria are abnormal in morphology, and there are no signs of mitochondrial fission; In yeast with N-terminally HA-tagged (right), mitochondrial morphology is abnormal, and there is no sign of mitochondrial fission;

[0038] Figure 2 In panel B, the horizontal axis represents wild-type yeast (WT) and Dnm1-labeled yeast, and the vertical axis represents the number of mitochondria per cell. Statistics show that the number of mitochondria per cell in wild-type yeast is 1 to 3, while the number of mitochondria per cell in Dnm1-labeled yeast is 1.

[0039] Figure 3 The scheme of using CRISPR technology to connect peptides to the surface of Dnm1 protein and the effect after connecting peptides are shown; Figure 3 Middle A: The left figure shows the structure of Dnm1 predicted at http: / / www.sbg.bio.ic.ac.uk / phyre2 / . The red and pink peptides are outward-facing loop sequences on the protein surface, which contain two predicted potential cleavage insertion sites, T680M681 (red arrow and red alphanumeric mark) and K586A587 (pink arrow and pink alphanumeric mark); the upper right figure is an enlarged portion of the dotted box in the left figure; the lower right figure shows different peptides connected to the positions pointed by the arrows in the upper right figure; yellow represents the Dnm1 peptide itself, red is the linker sequence, green is the GFP sequence, and purple is the HA tag; exogenous sequences were inserted into the K586 or T680 position of Dnm1 using CRISPR-Cas9 gene editing technology;

[0040] Figure 3 In B: The first row of images shows the Dnm1 target site Dnm1586 TH and Dnm1680 THConnected to GFP yeast, both have abnormal mitochondrial morphology and no signs of mitochondrial fission; the second row of images shows the Dnm1 target site Dnm1586 TH 、Dnm1680 TH The mitochondrial morphology of the two strains was abnormal, with no signs of mitochondrial fission. The third row of images shows Dnm1 target sites Dnm1586TH and Dnm1680TH linked to 1xHA yeast and wild-type yeast (WT). The mitochondrial morphology of the strains was similar to that of the wild-type, with obvious signs of mitochondrial fission (indicated by green arrows).

[0041] Figure 3 (C) Middle: The horizontal axis represents the wild-type yeast (WT) and Dnm1586 TH 、Dnm1680 TH Yeast with GFP, 5xHA, and HA peptide linked to the target sites in sequence. The vertical axis represents the number of mitochondria in each cell. Statistics show that wild-type yeast and Dnm1 680 TH HA, Dnm1586 TH The number of mitochondria in HA yeast cells ranged from 1 to 3, which was normal, with no significant difference in mitochondrial number. However, the number of mitochondria in yeast cells with GFP and 5xHA linked to the surface of Dnm1 was 1, which was abnormal.

[0042] Figure 4 The results of protein surface labeling on Dnm1 are shown. Figure 4 In A: wild-type yeast (WT), Dnm1680 TH HA / HA Nanobody-GFP and Dnm1586 TH Image of HA / HA Nanobody-GFP yeast. The green arrow indicates the obvious mitochondrial division. It shows the difference between wild-type yeast and Dnm1680. TH The mitochondrial morphology of HA / HA Nanobody-GFP yeast was consistent, and each cell mitochondria had multiple divisions, while Dnm1586 TH The mitochondrial morphology of HA / HA Nanobody-GFP yeast was abnormal, with fewer mitochondrial division sites;

[0043] Figure 4 In B: The horizontal axis represents different types of yeast, and the vertical axis represents the number of mitochondria in each cell; statistics show that wild-type yeast (WT) and Dnm1 680 TH The number of mitochondria in HA / HA Nanobody-GFP yeast was 1 to 3, which was normal, and there was no significant difference in the number of mitochondria; however, Dnm1586 THThe number of mitochondria in HA / HA Nanobody-GFP yeast decreased slightly, which may affect the function of Dnm1; the number of mitochondria in dnm1△, Dnm1-GFP and GFP-Dnm1 yeast was only 1, which is abnormal;

[0044] Figure 4 C is dnm1△, Dnm1 680 TH High-resolution live cell imaging of HA / HA Nanobody-GFP, GFP-Dnm1, and Dnm1-GFP yeast (taken every 1 minute), with blue arrows indicating mitochondrial fission; the left and right images are magnified images of the dotted box in the middle image, indicated by the gray dotted lines; the image shows Dnm1 680 TH HA / HA Nanobody-GFP yeast Dnm1680 TH HA mediated mitochondrial fission once between 0 and 1 min, and again between 7 and 8 min. No mitochondrial fission was observed in the other three yeast mitochondria.

[0045] Figure 4 D: The left picture shows Dnm1 680 without drug addition. TH HA / HANanobody-GFP imaging, the right image is Dnm1 680 after adding FCCP TH The dynamic changes of HA / HA Nanobody-GFP yeast were imaged by ultra-high resolution imaging, and the images were taken every 2 minutes. The image shows that before adding FCCP, a small amount of Dnm1 680 TH Localized on mitochondria, after adding FCCP, Dnm1 680 TH Dnm1 680 accumulates in large quantities on mitochondria. TH function, causing mitochondrial fragmentation;

[0046] Figure 5 This indicates that the C-terminal or N-terminal tagging of Mal3 affects its function; Figure 5A shows images of yeast cells containing mCherry-Atb2 (WT), mal3△ / mCherry-Atb2, Mal3-GFP / mCherry-Atb2, and GFP-Mal3 / mCherry-Atb2. The microtubules in mCherry-Atb2 yeast are longer than those in the other three yeast types and run through the entire cell. The microtubules in mal3△ / mCherry-Atb2 are significantly shortened, indicating that the absence of Mal3 causes microtubule shortening. The microtubules in Mal3-GFP / mCherry-Atb2 and GFP-Mal3 / mCherry-Atb2 yeast are also significantly shortened. This suggests that labeling Mal3 with GFP at both ends disrupts its function.

[0047] Figure 5 Middle B: The left panel shows a dotted statistical plot of microtubule length in yeast strains mCherry-Atb2 (WT), mal3△ / mCherry-Atb2, Mal3-GFP / mCherry-Atb2, and GFP-Mal3 / mCherry-Atb2. The right panel shows a statistical plot of the corresponding microtubule length distribution probability percentages. The left panel shows that the microtubule length of the mCherry-Atb2 (WT) strain is significantly different from that of the other strains. The right panel shows that the mCherry-Atb2 (WT) strain only accounts for about 20% of microtubules below 5 μm, while the other three strains account for more than 90%;

[0048] Figure 6 A scheme for attaching peptides to the surface of the Mal3 protein using CRISPR technology. The left image shows the Mal3 structure numbered 5m78, available at https: / / www.rcsb.org / . The red peptide is T680M681 (indicated by red arrows and red alphanumeric characters). The right image is an enlarged view of the dotted box in the left image.

[0049] Figure 7 The results of protein surface labeling on Mal3 are shown in Figure 2. Figure 7 A is the difference between mCherry-Atb2 yeast (WT) and Mal3 222 TH / HA NB-GFP / mCh-Atb2 yeast imaging; The figure shows the mCherry-Atb2 yeast (WT) and Mal3222 TH / HA NB-GFP / mCh-Atb2 yeast microtubule morphology is basically the same, and Mal3 222 TH Mal3 is correctly localized in HA NB-GFP / mCh-Atb2 yeast;

[0050] Figure 7 B is the mCherry-Atb2 yeast (WT) and Mal3 222TH / HA NB-GFP / mCherry-Atb2 yeast microtubule number dot plot; mCh-Atb2 yeast (WT) and Mal3 222 TH / HA NB-GFP / mCh-Atb2 yeast had the same number of microtubules and no difference;

[0051] Figure 7 In C, the left image shows mCherry-Atb2 yeast (WT) and Mal3 222 TH / HA NB-GFP / mCh-Atb2 yeast microtubule length dot plot; mCherry-Atb2 yeast (WT) and Mal3 222 TH The microtubule lengths of yeast strains with HA NB-GFP and mCh-Atb2 are basically the same. The right figure shows the probability distribution percentage of microtubule lengths corresponding to the left figure. The microtubule length distribution probabilities of the two strains are basically the same.

[0052] Figure 7 In D, (1), (2), (3), and (4) are mCherry-Atb2 yeast (WT) and Mal3 222 TH / HA NB-GFP / mCherry-Atb2 yeast microtubule growth rate, contraction rate, residence time at the cell end, and maximum length within 10 minutes are dotted statistically, which are four basic microtubule dynamics parameters; Statistics show that mCherry-Atb2 yeast (WT) and Mal3222 TH The basic parameters of the four microtubule dynamics in yeast strains with / HA NB-GFP / mCh-Atb2 were basically consistent;

[0053] Figure 7 E is Mal3 222 TH / HA NB-GFP / mCh-Atb2 yeast high-resolution live cell imaging; the middle figure shows the dynamic changes of microtubules and Mal3 within 10 minutes. The left and right figures are magnified images of the blue box in the middle figure, indicated by the gray dotted line; the red arrow indicates the process of Mal3 moving along the microtubule to the end of the microtubule, the blue arrow indicates the process of microtubule disappearing from the microtubule and Mal3 disappearing at the end of the microtubule, and the yellow arrow indicates that the microtubule remains stable; the microtubule remains stable at the point indicated by the yellow arrow, indicating that Mal3 can maintain microtubule stability, and the blue arrow indicates the disappearance of Mal3, indicating that the disappearance of Mal3 may cause microtubule instability and shortening of microtubules. At the intersection of the red and blue arrows, Mal3 returns to the end of the microtubule and the microtubule begins to grow;

[0054] Figure 8A scheme for attaching peptides to the surface of the Nda3 protein using CRISPR technology is shown. The left image shows the Nda3 structure in the 5mjs complex structure provided by https: / / www.rcsb.org / . The red peptide is Y36 H37 (red arrows and red letters and numbers). The right image is an enlarged view of the dotted box in the left image.

[0055] Figure 9 The results of protein surface labeling on Nda3 are shown in Figure 2. Figure 9 The left side of the middle dashed line in A shows an image of microtubules labeled using the traditional method for fission yeast, and the right side shows an image of microtubules labeled using the new method. The image on the right side of the middle dashed line shows that the new method can label microtubules well, and compared with the traditional method (left side of the dashed line), the new method has a better effect on labeling microtubules.

[0056] Figure 9 B shows the dotted statistical graph of the number of microtubules in each yeast cell labeled by the traditional method and the new method, showing that the number of microtubules in the four yeasts is basically the same;

[0057] Figure 9 In C, the left and right figures are dotted statistical graphs of microtubule lengths labeled by the traditional method and the new method, and statistical graphs of the probability percentage of microtubule length distribution, respectively. The left figure shows that the microtubule lengths of GFP-Atb2 yeast are significantly different from those of the other three yeasts, with longer microtubules; the right figure shows that the probability of longer microtubules in GFP-Atb2 yeast is higher than that of the other three yeasts;

[0058] Figure 9 D shows the dynamic changes of microtubules labeled by the traditional method and the new method within 190 seconds, indicating that the new method can label microtubules well.

[0059] Figure 9 In E, (1)(2)(3)(4) show the dotted statistical graphs of the growth rate, contraction rate, residence time at the cell end, and maximum length that can be achieved of the microtubules labeled by the traditional method and the microtubules labeled by the new method within 190 seconds, which are the four basic microtubule dynamic parameters; statistics show that the contraction rate of GFP-Atb2 yeast microtubules and the residence time of microtubules at the cell end are significantly different from those of the other three yeast microtubules, that is, GFP-Atb2 yeast microtubules contract faster and the microtubules stay at the cell end longer, while the other two dynamic parameters of the four bacteria are basically consistent. DETAILED DESCRIPTION

[0060] The present invention discloses a composition and method for labeling or tracing a target protein. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0061] (1) The N- and C-termini of proteins such as Dnm1, Mal3, and Nda3 cannot be labeled, as labeling would affect protein function. To address this problem, we developed a novel method described in this invention that allows for labeling of these proteins without affecting their function.

[0062] (2) The special design is the key point of the present invention. We refer to the protein structure information to find the rings that are fully exposed on the surface of the protein, and perform hydrophobicity and hydrophilicity analysis on the amino acids on the rings. Then, we use CRISPR-Cas9 gene editing technology to insert small tags recognized by nanobodies at the sites in the protein surface rings, and then use the corresponding fluorescent protein fused nanobody (such as nanobody-GFP) to identify and locate the target protein that is surface-labeled with the nanobody recognizing the small tag (see Figure 1 ).

[0063] Design of a new method for fluorescent labeling of protein surfaces

[0064] In order to overcome the problem of not being able to label the target protein at the C-terminus or N-terminus, we have developed a new target protein labeling method. Figure 1 shown.

[0065] The new method design is described in detail as follows:

[0066] (1) Find the loop sequence facing outward on the surface of the target protein. You can obtain the resolved target protein structure from https: / / www.rcsb.org / , or http: / / www.sbg.bio.ic.ac.uk / phyre2 / Analyze the structure of the unsolved target protein using the alphafold and https: / / www.alphafold.ebi.ac.uk / websites. Next, identify loop sequences on the target protein surface that face outwards based on the target protein structure. These loop sequences should avoid being within the interaction interface between the target protein and other interacting proteins.

[0067] (2) Selection of insertion sites within the loop sequence. According to the website https: / / dokhlab.med.psu.edu / spell / login.php, obtain multiple possible cleavage insertion sites for the target protein. If the predicted cleavage insertion site is within the loop sequence identified in (1), the cleavage insertion site is the target site. If the predicted cleavage insertion site does not fall within the loop sequence identified in (1), multiple sites within the identified loop sequence can be directly tried and verified through subsequent experiments.

[0068] (3) Modify the target protein and insert a Nanobody recognition small tag with flexible amino acid connecting peptides at both ends into the target protein target site. The CRISPR-Cas9 system can be used to connect the Nanobody recognition small tag with flexible amino acid connecting peptides at both ends to the target protein target site. The hydrophilic connecting peptides at both ends of the small tag can fully expose the small tag on the protein surface. The total number of amino acids in the Nanobody recognition small tag and the flexible amino acid connecting peptides at both ends can be controlled between 30 and 50, which is very small and has good flexibility. This can largely avoid folding errors caused by interference from the inserted peptides during protein folding in vivo.

[0069] (4) Constructing a fusion expression vector of nanobody and GFP. Through molecular cloning methods, the nanobody sequence was connected to the green fluorescent protein (GFP) gene sequence in the vector to construct the Nanobody-GFP plasmid.

[0070] (5) Target protein tracking. The Nanobody-GFP plasmid is transfected or transformed into the cells expressing the Nanobody recognition tag in (3) and the localization of the target protein and its dynamic localization changes are observed using a fluorescence microscope.

[0071] In some preferred embodiments of the present invention, given the ease of genetic manipulation in fission yeast, we selected fission yeast as a model organism and used previously published HA-tagged nanobodies as representatives (various types of nanobodies and their corresponding specific recognition tags can be used) to test the feasibility of the above-mentioned new method. Following the above-mentioned design, we first used CRISPR-Cas9-based gene editing technology to attach HA tags with flexible amino acid linker peptides at both ends within the protein sequences of Dnm1 (mitochondrial fission regulatory protein), Mal3 (microtubule plus end binding protein), and Nda3 (β-tubulin). We then used HANanobody-GFP to successfully trace the surface-labeled HA-tagged Dnm1, Mal3, and Nda3 proteins. Furthermore, this method can also utilize different nanobodies and their specific recognition tags to achieve diverse protein tracing.

[0072] Based on the above, since the traditional fluorescent labeling method is to connect fluorescent proteins to the N-terminus or C-terminus of the target protein, but some proteins are not suitable for N-terminal or C-terminal labeling, some researchers choose to directly insert fluorescent proteins into certain proteins (without a specific solution). We also tried this method, and we connected GFP and 5xHA to the Dnm1 protein sequence (see Figure 3 A) Through microscopic imaging, we found that this method does not work for Dnm1. Figure 3 After the two peptides in A are inserted into Dnm1, Dnm1 does not have normal biological functions ( Figure 3 B, C), this may be because GFP and 5xHA are too large and inserted into the interior of Dnm1, affecting the spontaneous folding of Dnm1 and thus affecting the function of Dnm1.

[0073] We successfully tagged three proteins, Dnm1, Mal3, and Nda3, using a small HA tag with flexible amino acid linker peptides at both ends, attached to their surfaces (within their protein sequences). This peptide, GGSGGSGGSGGSGGSGGSYPYDVPDYAGGSGGSGGSGGSGGSGGS (shown in SEQ ID No. 1), consists of only 45 amino acids, with six repeating GGS amino acids at each end forming a flexible linker sequence. Because the repeating GGS amino acids (the main component of the peptide) have small side chains and are relatively hydrophilic (G hydrophobicity parameters of -0.4 and S of -0.8), they easily protrude the hydrophilic YPYDVPDYA (HA) into the aqueous phase during protein folding, forming a hairpin structure. These 45 amino acids, when attached to the target protein at the appropriate position (outward-facing loops on the protein surface), easily protrude into the aqueous phase, isolating the HA from the protein surface. This significantly reduces the influence of the linker peptide on the folding of the target protein backbone. Our experiments also proved the rationality of our idea. We also proved that peptides can be connected to the surface of proteins (inside protein sequences), but it is related to the size, properties and connection position of the connected peptides, such as Figure 3 A, B, C.

[0074] The present invention not only provides a solution for discovering the position of connecting peptide segments inside proteins, but also provides a very small and flexible connecting peptide segment, and provides a new strategy for locating and tracking surface-labeled target proteins using nanobody-fluorescent protein.

[0075] In the target protein labeling or tracing composition and method provided by the present invention, the raw materials and reagents used can be purchased from the market.

[0076] The present invention will be further described below in conjunction with the embodiments:

[0077] Example 1 Dnm1 tracing

[0078] 1.1 Fluorescent labeling of the N or C terminus of Dnm1 affects its function

[0079] In fission yeast, Dnm1 is the only known protein that directly mediates mitochondrial fission. Our research has found that wild-type yeast mitochondria typically form a complex network structure, with multiple mitochondria per cell. When Dnm1 is missing in fission yeast, mitochondria do not divide, their mitochondrial morphology is abnormal, and their number is only one (see Figure 2 A); the statistical diagram of the number of mitochondria in each cell is Figure 2 B. When the C-terminus and N-terminus of Dnm1 were labeled with GFP or HA, the mitochondrial phenotype was the same as that of mitochondria lacking Dnm1 (see Figure 2 A. Figure 2 B) When the GFP tag was replaced with mCherry or tdTomato, two red fluorescent protein tags, the mitochondrial phenotype was the same as that of the mitochondria when Dnm1 was missing ( Figure 2 B) This indicates that labeling Dnm1 at its C-terminus or N-terminus will affect its function. Therefore, to trace Dnm1, we need to employ our new protein surface fluorescent labeling method.

[0080] 1.2 Design of Dnm1 surface fluorescent labeling and evaluation of whether surface labeling affects mitochondrial morphology and dynamics

[0081] Since the tagging of both ends of Dnm1 affects the protein function, we tried to tag Dnm1 using a new method. http: / / www.sbg.bio.ic.ac.uk / phyre2 / The predicted structure on the protein surface was used to confirm the loop sequence facing outwards on the surface. Then, multiple possible cleavage insertion sites of Dnm1 were obtained according to the website https: / / dokhlab.med.psu.edu / spell / login.php. Among them, two predicted cleavage insertion sites were located on the protein surface facing outwards on the loop sequence, namely K586A587 and T680M681 (see Figure 3 A).

[0082] We named the target site "protein name". TH ". For example, if the target site is between T680 and M681, it is uniformly named Dnm1 680 TH We refer to proteins with peptides attached to the outward-facing loop sequence on the protein surface as "protein name-attachment position" TH For example, if an HA peptide with flexible amino acid linker peptides at both ends is connected at the positions of Dnm1 T680 and M681 (target site), it will be named Dnm1 680 TH HA.

[0083] We used CRISPR-Cas9 gene editing technology to TH 、Dnm1 680 TH The target sites were connected to GFP, 5xHA and HA respectively. Through imaging, we found that Dnm1 586 TH 、Dnm1 680 TH The yeast mitochondria in which GFP and 5xHA were linked to the target site had abnormal mitochondrial morphology, did not divide, and had only one mitochondrion (see Figure 3 B, C). However, Dnm1586 TH 、Dnm1 680 TH The yeast cells with HA linked to the target site had normal mitochondrial morphology and normal mitochondrial number, that is, the mitochondria could divide normally (see Figure 3 B, C).

[0084] 1.3 HA Nanobody-GFP localization and tracing of surface-labeled HA Dnm1

[0085] By molecular cloning, the HA Nanobody sequence was connected to the green fluorescent protein (GFP) gene sequence in the vector to construct the HA Nanobody-GFP plasmid. TH HA and Dnm1 680 TH HA strains were transformed with HA Nanobody-GFP plasmid, so that HA Nanobody-GFP was integrated into the yeast genome, and then HA Nanobody-GFP was stably expressed in both strains. Microscopic imaging revealed that Dnm1 680 TH The mitochondrial morphology of HA / HA Nanobody-GFP bacteria is basically the same as that of wild type, and the number of mitochondria is also consistent with that of wild type yeast. TH HA can be recognized and localized by HANanobody-GFP (see Figure 4 A, B). We used high-resolution live cell imaging to investigate the expression of dnm1△ and Dnm1 680 TH HA / HA Nanobody-GFP, Dnm1-GFP, and GFP-Dnm1 strains were imaged and it was found that Dnm1 680 TH HA can mediate mitochondrial fission (see Figure 4C) Dnm1 has the function of dividing mitochondria. The mitochondrial oxidative phosphorylation uncoupler FCCP can promote the recruitment of Dnm1 to mitochondria. If Dnm1 functions normally, the Dnm1 enriched in mitochondria after FCCP treatment of yeast strains will cause a large number of mitochondria to break and show a fragmented morphology. Therefore, we used FCCP to treat Dnm1680 TH HA / HA Nanobody-GFP strain to further verify Dnm1 680 TH After FCCP treatment, we found that Dnm1 680 TH Dnm1 680 in HA / HA Nanobody-GFP strain TH After a certain reaction time, HA accumulates in large quantities on mitochondria and plays a role in fragmenting mitochondria (see Figure 4 D). It can be seen that Dnm1680 TH HA has normal biological functions, and our new protein surface fluorescent labeling method can locate and observe it without affecting the function of Dnm1.

[0086] Example 2 Mal3 tracing

[0087] Mal3 is a microtubule plus-end binding protein in fission yeast that maintains microtubule stability.

[0088] 2.1 Fluorescent labeling of the N or C terminus of Mal3 affects its function

[0089] We found that C-terminal or N-terminal tagging of Mal3 resulted in very short microtubules, similar to the microtubule phenotype of cells lacking Mal3 (see Figure 5 A, B). This suggests that GFP tagging of the C- or N-terminus of Mal3 affects Mal3 function. When Mal3 is nonfunctional or impaired, microtubules become unstable and prone to premature depolymerization. Consequently, microtubules are unable to reach wild-type length, resulting in a shorter microtubule phenotype.

[0090] 2.2 Design of Mal3 surface fluorescent marker

[0091] Since the labeling of Mal3 at both ends affects the protein function, we tried to use a new method to label Mal3. First, we obtained the resolved structure of fission yeast Mal3 (PDB: 5m79) according to https: / / www.rcsb.org / (see Figure 6). Based on the Mal3 structure, the only surface-outward-facing loop sequence was found. Then, the potential cleavage insertion site of Mal3 was predicted according to the website https: / / dokhlab.med.psu.edu / spell / login.php, but no predicted cleavage site was found to fall within the only surface-outward-facing loop sequence identified. Therefore, we analyzed the amino acids and amino acid positions of the Mal3 surface-outward-facing loop sequence and selected an HA peptide segment with flexible amino acid linker peptide segments at both ends between the most fully exposed T222 and S223 (see Figure 6 ).

[0092] 2.3 HA Nanobody-GFP localization and tracking of Mal3 and evaluation of whether surface labeling affects its microtubule regulation function

[0093] We used CRISPR-Cas9 gene editing technology to insert an HA peptide with flexible amino acid linker peptides at both ends between Mal3 T222 and S223. TH In HA yeast, HA nanobody-GFP was used to target Mal3 222 TH HA was used for localization tracking. High-resolution live cell imaging analysis revealed that HAnanobody-GFP could well recognize Mal3 surface-labeled with HA, and the microtubule length was normal (see Figure 7 A, C). We compared mCherry-Atb2 yeast with Mal3 222 TH The number of microtubules in HA / HA nanobody-GFP yeast is basically the same (see Figure 7 B) In addition, we also compared the mCherry-Atb2 yeast with Mal3 222 TH HA / HA nanobody-GFP yeast microscopic dynamic parameters (see Figure 7 D); The four microtubule dynamic parameters of the two yeast strains were basically consistent. This shows that the new method of labeling Mal3 does not affect the function of Mal3. We further performed high-resolution live cell imaging observations and analysis and found that when Mal3 disappeared from the microtubule end, the microtubule was unstable and shortened; when Mal3 re-bound to the microtubule end, the microtubule remained stable and grew (see Figure 7 E) This shows that our new protein surface fluorescent labeling method can effectively locate and track Mal3 without affecting the biological function of Mal3.

[0094] Example 3 Nda3 localization observation

[0095] Microtubules are composed of polymers of α-tubulins and β-tubulins. In fission yeast, α-tubulin Nda2 and β-tubulin Nda3 are essential genes, and labeling at both ends of these proteins causes lethality. Consequently, most researchers have been able to visualize microtubules only by labeling the N-terminus of the non-essential α-tubulin Atb2 gene (the C-terminus cannot be labeled). We utilized the novel method developed in this paper to label β-tubulin Nda3 and assess changes in microtubule function.

[0096] 3.1 Design of a new method for labeling β-tubulin

[0097] First, we obtained the solved fission yeast Nda3 complex structure (PDB: 5mjs) from https: / / www.rcsb.org / ; according to the Nda3 structure (see Figure 8 ), we searched for the target protein’s surface-facing loop sequence at the interface where the target protein does not interact with its interacting proteins. Then, according to the website https: / / dokhlab.med.psu.edu / spell / login.php, we obtained multiple potential cleavage insertion sites for the target protein. Among them, one cleavage site located on the protein surface-facing loop sequence is Y36H37. Therefore, we chose to connect the HA peptide with flexible amino acid linker peptides at both ends between Y36 and H37 of Nda3 (see Figure 8 ).

[0098] 3.2 Localization and tracking of Nda3 using HA Nanobody-GFP and assessing whether surface labeling affects microtubule dynamics

[0099] We used CRISPR-Cas9 gene editing technology to connect an HA peptide with flexible amino acid linker peptides at both ends between Y36 and H37 of Nda3. Then, HA Nanobody-GFP and HA Nanobody-yMscarlet plasmids were transformed into Nda3 36 TH HA yeast, and then Nda336 through HA nanobody-GFP and HA Nanobody-yMscarlet TH We successfully labeled microtubules with two colors, and the imaging effect was better than that of microtubules labeled with traditional methods (see Figure 9 A). The results of microtubule number statistics showed that the number of microtubules in the four yeasts was basically the same (see Figure 9 B). In addition, the microtubule length measurement results showed that the microtubule length of GFP-Atb2 was significantly different from that of the other three bacteria (see Figure 9 C). We further performed high-resolution live cell imaging observations and the results showed that the microtubules of the four cells could shrink and grow normally (see Figure 9D). We measured the microtubule dynamics parameters of the four strains and the results showed that the contraction rate of GFP-Atb2 microtubules was significantly different from that of the other three strains. The contraction rate of GFP-Atb2 microtubules was faster, and the GFP-Atb2 microtubules stayed at the cell end longer (see Figure 9 E). mCherry-Atb2 microtubules are the most commonly used and recognized method for labeling microtubules in academia. However, the phenotypes of GFP-Atb2 microtubules and mCherry-Atb2 microtubules are not completely consistent. This suggests that there may be some problems with the Atb2 N-terminal labeling that affect microtubules. The dynamic parameters of microtubules labeled with the new method are basically consistent with those of mCherry-Atb2 microtubules in terms of number, length, and dynamics (see Figure 9 B, C, E). This shows that the new method not only does not affect the function of Nda3, but also can effectively label microtubules. In particular, the green microtubules labeled by the new method are more prominent than the traditional green microtubule labeling method.

[0100] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention. Sequence Listing <110> University of Science and Technology of China <120> Target protein labeling or tracing composition and method <130> MP21024746 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 45 <212> PRT <213> Artificial Sequence <400> 1 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 15 Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Gly Gly Ser Gly Gly 20 25 30 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 35 40 45

Claims

1. A target protein labeling or tracing method, characterized in that: The steps include: Step 1: Obtaining the outward-facing loop sequence of the target protein; Step 2: inserting a small tag with flexible amino acid linker peptides at both ends into the loop sequence; Step 3: Use a small tag nanobody fused with a fluorescent protein corresponding to a small tag with flexible amino acid linker peptides at both ends to identify, locate and track the target protein; When the target protein is Dnm1 from fission yeast, the insertion position is between T680 and M681; or When the target protein is Mal3 from fission yeast, the insertion position is between T222 and S223; or When the target protein is Nda3 from fission yeast, the insertion position is between Y36 and H37; The small tag with flexible amino acid connecting peptide segments at both ends is an HA tag; the small tag nanobody fused with fluorescent protein is an HA nanobody.

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