CN114705846A - Labeling or tracking compositions and methods of target proteins - Google Patents

Abstract

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. The invention firstly analyzes the structure of the target protein and determines the loop sequence of the protein surface outward orientation, then inserts small labels (such as HA labels) with flexible amino acid connecting peptide segments at two ends into the loop sequence by a molecular cloning method, and finally expresses the small label nano antibody of the fusion fluorescent protein, thereby realizing the intracellular tracing of the target protein by utilizing the characteristic that the nano antibody specifically identifies the target protein surface label.

Description

Labeling or tracking compositions and methods of target proteins

technical field

The present invention relates to the field of protein labeling or tracing, in particular to a target protein labeling or tracing composition and method.

Background technique

In order to analyze the dynamic localization of proteins in cells, it is necessary to fluorescently label the target proteins and use live-cell fluorescence microscopy to image them. 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 proteins 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 protein structure will be destroyed after connecting the fluorescently labeled protein; the C-terminus or N-terminus of the target protein may also interact with other proteins, and after connecting the fluorescently labeled protein, it will Destruction of protein interactions; the C-terminus or N-terminus of the target protein may mediate direct interactions with cell substructures or organelles, and the connection of fluorescently labeled proteins will affect the accurate positioning of the target.

The traditional fluorescent labeling method is to directly link fluorescently labeled proteins at the C-terminus or N-terminus of the target protein, but this method is not suitable for all proteins. Existing protocols insert fluorescently labeled proteins directly inside the target protein. However, directly inserting the fluorescently labeled protein into the target protein often affects the function of the target protein. Because fluorescently labeled proteins are mostly globular proteins with nearly 200 amino acids, the insertion of this larger fluorescently labeled protein may affect the structural stability of the target protein, thereby causing the target protein to lose its function.

In summary, fluorescent labeling of target proteins by traditional methods may disrupt the protein structure and its intracellular localization, thereby affecting the function of the protein and even normal cell proliferation. Therefore, it is of great practical significance to provide a new method for protein surface fluorescent labeling.

SUMMARY OF THE INVENTION

In view of this, the present invention does not label the fluorescent protein at the C-terminus or N-terminus of the target protein, but realizes the fluorescent labeling of the target protein by referring to the protein structure and connecting the tagged protein on the surface of the protein (in the middle of the protein sequence).

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

In the first aspect, the present invention provides the application of a small tag with a flexible amino acid linking peptide segment at both ends and a small tag nanobody fused to a fluorescent protein in the labeling or tracing of a target protein.

In some specific embodiments of the present invention, the small tags with flexible amino acid linking peptides at both ends include HA tags; the small tag nanobodies fused to fluorescent proteins include HA nanobodies.

In some specific embodiments of the present invention, the insertion position of the small tag with flexible amino acid linking peptides at both ends includes any position of the loop sequence facing outward on the surface of the target protein.

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

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

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

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

In the second aspect, the present invention also provides a target protein labeling or tracking composition, including small tags with flexible amino acid linking peptides at both ends and small tag nanobodies fused to fluorescent proteins.

In some specific embodiments of the present invention, the small tags with flexible amino acid linking peptides at both ends include HA tags; the small tag nanobodies fused to fluorescent proteins include HA nanobodies.

In some specific embodiments of the present invention, the insertion position of the small tag with flexible amino acid linking peptides at both ends includes any position of the loop sequence facing outward on the surface of the target protein.

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

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

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

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

In a third aspect, the present invention also provides the application of the labeling or tracking composition in the preparation of a kit or device for labeling or tracking a target protein.

In a fourth aspect, the present invention also provides a kit or device comprising an acceptable carrier or adjuvant in the section of the labeling or tracking composition.

In a fifth aspect, the present invention also provides a target protein labeling or tracking method based on the labeling or tracking composition or the kit, comprising the following steps:

Step 1: obtain the loop sequence facing the surface of the target protein;

Step 2: inserting a small tag with flexible amino acid linking peptides at both ends at the position of the loop sequence;

Step 3: Identify and locate and track the target protein by using the small tag nanobody of the fusion fluorescent protein corresponding to the small tag with flexible amino acid linking peptides at both ends.

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

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

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

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

In order to overcome the problem that the target protein cannot be labeled at the C-terminus or the N-terminus, the present invention provides a brand-new target protein labeling method. First, we analyze the structure of the target protein and determine the loop sequence facing the surface of the protein, then insert a small tag (such as HA tag) with flexible amino acid linking peptides in the middle of the loop sequence by molecular cloning method, and finally express the fusion Small-labeled nanobodies of fluorescent proteins (such as HA nanobody) can use the characteristics of nanobodies to specifically recognize the surface tags of target proteins to achieve intracellular tracking of target proteins.

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

(1) The N- and C-termini of Dnm1, Mal3, Nda3, etc. cannot be labeled, which will affect the protein function after labeling. To address the issue of fluorescent labeling of this protein, we have developed the novel methods described in the present invention to enable labeling of this type of protein without affecting these functions.

(2) Special design is the key point of the present invention. By referring to the protein structure information, we searched for loops that were fully exposed on the surface of the protein, and analyzed the hydrophobicity and hydrophilicity of amino acids on the loops. Then, using CRISPR-Cas9 gene editing technology, we inserted nanobodies into the loops on the surface of the protein. The identified small tag, and then use the corresponding fluorescent protein fusion nanobody (eg: nanobody-GFP) to identify and locate and track the target protein of the surface labeled nanobody to recognize the small tag (see Figure 1).

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art.

Fig. 1 shows the experimental process implemented in the present invention; wherein, (1) shows the use of CRISPR technology to connect the outer-facing loop sequence position of the endogenous target protein surface to connect a Nanobody (Nanobody) identification tag with flexible amino acid linking peptides at both ends ; (2) shows that the nanobody sequence is connected with a green fluorescent protein (GFP)-containing vector to obtain a nanobody-green fluorescent protein (abbreviated NB-GFP) plasmid; (3) shows that the NB-GFP plasmid was transformed or transfected into The surface of the endogenous target protein is connected to the cell with the Nanobody-recognized small tag, and the plasmid expresses NB-GFP stably; (4) In the end, NB-GFP specifically recognizes the Nanobody-recognized small tag to realize the tracking of the target protein;

Figure 2 shows that the C-terminal or N-terminal labeling of Dnm1 affects its function; in Figure 2A: the top row of images are wild-type (WT) yeast (left) and dnm1 knockout (dnm1△) yeast (right). ) imaging image, Mitotracker is a mitochondrial dye, which can stain mitochondria in red. In wild-type yeast, the mitochondrial shape is a tubular network with obvious breaks (the green arrow points); in dnm1 gene-deficient yeast, mitochondria have no fission. In the middle row and the bottom row, the images of the two ends of Dnm1 are labeled; the images in the middle row are the images of the labeled GFP at both ends of Dnm1, and the yeast with GFP labeled at the C end of Dnm1 (left), Dnm1- GFP is located in mitochondria, but the mitochondrial morphology is abnormal, and there is no sign of mitochondrial fission; in the yeast with Dnm1 N-terminal labeled GFP (right), GFP-Dnm1 is located in mitochondria, but the mitochondrial shape is abnormal, and there is no sign of mitochondrial fission; the bottom image shows Imaging image of HA labeled at both ends of Dnm1, in the yeast with HA labeled at the C-terminus of Dnm1 (left), the mitochondrial morphology is abnormal, and there is no sign of mitochondrial fission; in the yeast labeled with HA at the N-terminus of Dnm1 (right), the mitochondrial morphology is abnormal, and the mitochondria have no fission. sign;

In Figure 2B: the abscissa is the wild-type yeast (WT) and the labeled yeast at both ends of Dnm1, and the ordinate is the number of mitochondria in each cell; statistics show that the number of mitochondria per cell of the wild-type yeast is 1-3, while The number of mitochondria in each cell of yeast labeled at both ends of Dnm1 is 1;

Figure 3 shows the scheme of using CRISPR technology to connect peptides on the surface of Dnm1 protein and the effect after connecting peptides; among them, in Figure 3A: the left picture is Dnm1 at http://www.sbg.bio.ic.ac.uk/phyre2 / The predicted structure above, the red and pink peptides are the outer surface facing loop sequences of the protein, which contain T680M681 (red arrows and red alphanumeric markers) and K586A587, (pink arrows and pink alphanumeric markers) two predicted potential splits Insertion site; the upper right picture is the enlarged part of the dotted box in the left picture; the lower right picture shows the different peptides connected by the arrows in the upper right picture; among them, the yellow indicates the Dnm1 self-peptide, the red is the linker sequence, and the green is the GFP sequence , the purple is the HA tag; the exogenous sequence is inserted into the K586 or T680 position of Dnm1 by using CRISPR-Cas9 gene editing technology;

In Figure 3B: The first row of imaging images shows that Dnm1 target sites Dnm1586 ^TH and Dnm1680 ^TH are connected to GFP yeast, both of which have abnormal mitochondrial morphology and no signs of mitochondrial fission; the second row of imaging images are Dnm1 target sites Dnm1586 ^TH , Dnm1680 ^TH is connected to 5xHA yeast, both of which have abnormal mitochondrial morphology and no signs of mitochondrial fission; the third row of images shows that Dnm1 target sites Dnm1586TH and Dnm1 680TH are connected to 1xHA yeast and wild-type yeast strain (WT), and the mitochondrial morphology of the strain is the same as that of wild-type yeast strain (WT). Type mitochondria are similar, and the mitochondria have obvious signs of division (green arrows);

In Figure 3(C): the abscissa is the wild-type yeast (WT) and the yeast with the target sites of Dnm1586 ^TH and Dnm1680 ^TH connected to GFP, 5xHA and HA peptides in turn, and the ordinate is the number of mitochondria in each cell; statistics It shows that the number of mitochondria in wild-type yeast and Dnm1 680 ^TH HA and Dnm1586 ^TH HA yeast cells is 1 to 3, the number of mitochondria is normal, and there is no significant difference in the number of mitochondria; while the number of mitochondria per cell of yeast with GFP and 5xHA attached to the surface of Dnm1 is 1, the number of mitochondria is abnormal;

Figure 4 shows the effect of protein surface labeling method applied to Dnm1; among them, in Figure 4A: wild-type yeast (WT), Dnm1680 ^TH HA/HA Nanobody-GFP and Dnm1586 ^TH HA/HA Nanobody-GFP yeast imaging images, green The arrows indicate the obvious mitochondrial fission; it shows that the mitochondrial morphology of wild-type yeast is consistent with that of Dnm1680 ^TH HA/HA Nanobody-GFP yeast, and the mitochondria of each cell have multiple fissures, while the mitochondrial morphology of Dnm1586 ^TH HA/HA Nanobody-GFP yeast Abnormal, less mitochondrial fission;

In Figure 4B: the abscissa is the different types of yeast, and the ordinate is the number of mitochondria in each cell; statistics show that the number of mitochondria in wild-type yeast (WT) and Dnm1 680 ^TH HA/HA Nanobody-GFP yeast is 1-3 , the number of mitochondria was normal, and there was no significant difference in the number of mitochondria; however, the number of mitochondria in Dnm1586 ^TH HA/HA Nanobody-GFP yeast decreased slightly, and the function of Dnm1 may be affected; the number of mitochondria in dnm1△, Dnm1-GFP and GFP-Dnm1 yeast is 1, the number is abnormal;

Figure 4C is a high-resolution live-cell imaging image of dnm1△, Dnm1 680 ^TH HA/HA Nanobody-GFP, GFP-Dnm1 and Dnm1-GFP yeast (taken every 1 minute), blue arrows indicate mitochondrial division; left The picture and the right picture are the enlarged part of the ^dashed box in the middle picture, which is indicated by the gray ^dashed line. Mitochondrial fission was mediated again within 8 min, and no mitochondrial fission was observed in the mitochondria of the other three yeasts;

Figure 4D: The left picture is the image of Dnm1 680 ^TH HA/HANanobody-GFP without drug added, and the right picture is the dynamic change of Dnm1 680 ^TH HA/HA Nanobody-GFP yeast ultra-high resolution imaging after adding FCCP. Shot once every 2 minutes; the figure shows that in the same cell, before adding FCCP, a small amount of Dnm1 680 ^TH was localized on mitochondria. After adding FCCP, ^Dnm1 680 ^TH accumulated a lot on mitochondria. fragment mitochondria;

Figure 5 shows that the C-terminal or N-terminal labeling of Mal3 affects its function; among them, Figure 5A shows that mCherry-Atb2 (WT), mal3Δ/mCherry-Atb2, Mal3-GFP/mCherry-Atb2 and GFP-Mal3/mCherry-Atb2 yeast The microtubule length of mCherry-Atb2 yeast is longer than the other three yeast microtubules and runs through the whole cell; the microtubules of mal3△/mChery-Atb2 are obviously shortened, indicating that the deletion of Mal3 will cause the shortening of microtubules; Mal3-GFP /mCherry-Atb2, GFP-Mal3/mCherry-Atb2 yeast microtubules are also significantly shortened; this shows that the labeling of GFP at both ends of Mal3 will destroy the function of Mal3;

In Figure 5B: the left picture is the dotted statistics of yeast microtubule length of mCherry-Atb2 (WT), mal3Δ/mCherry-Atb2, Mal3-GFP/mCherry-Atb2 and GFP-Mal3/mCherry-Atb2, and the right picture is the left The figure corresponds to the percentage of probability distribution of microtubule length; the left figure shows that the microtubule length of mCherry-Atb2 (WT) strain is significantly different from that of other strains, and the right figure shows that the microtubules below 5 microns mCherry-Atb2 (WT) strains account for only about 20%, while the remaining three strains account for more than 90%;

Figure 6 shows the scheme of connecting peptides on the surface of Mal3 protein by CRISPR technology; the left picture is the Mal3 structure numbered 5m78 provided by https://www.rcsb.org/; the red peptide is T680M681 (red arrows and red letters and numbers) mark), the right picture is the enlarged part of the dotted box in the left picture;

Figure 7 shows the effect of protein surface labeling method applied to Mal3; wherein, Figure 7A is the imaging image of mCherry-Atb2 yeast (WT) and Mal3 222 ^TH /HA NB-GFP/mCh-Atb2 yeast; the figure shows mCherry-Atb2 yeast The microtubule morphology of bacteria (WT) was basically the same as that of Mal3222 ^TH /HA NB-GFP/mCh-Atb2 yeast, and Mal3 was located correctly in Mal3 222 ^TH HA NB-GFP/mCh-Atb2 yeast;

Figure 7B is a dotted graph of the number of microtubules in mCherry-Atb2 yeast (WT) and Mal3 222 ^TH /HA NB-GFP/mCherry-Atb2 yeast; mCh-Atb2 yeast (WT) and Mal3 222 ^TH /HA NB- The number of GFP/mCh-Atb2 yeast microtubules was the same, but there was no difference;

In Figure 7C, the left graph is the microtubule length plot of mCherry-Atb2 yeast (WT) and Mal3 222 ^TH /HA NB-GFP/mCh-Atb2 yeast; mCherry-Atb2 yeast (WT) and Mal3 222 ^TH /HA NB-GFP/mCh-Atb2 yeast microtubule lengths are basically the same; the right figure is the percentage statistics of the microtubule length distribution probability corresponding to the left figure; the microtubule length distribution probabilities of the two are basically the same;

In Figure 7D, (1), (2), (3), (4) are mCherry-Atb2 yeast (WT) and Mal3 222 ^TH /HA NB-GFP/mCherry-Atb2 yeast microtubules within 10 minutes, respectively The growth rate, shrinkage rate, residence time at the cell end, and the maximum length that can be reached are the four basic parameters of microtubule dynamics. Statistics show that mCherry-Atb2 yeast (WT) and Mal3222 ^TH /HA NB- The four basic parameters of microtubule dynamics in GFP/mCh-Atb2 yeast are basically the same;

Figure 7E is a high-resolution live cell imaging image of Mal3 222 ^TH /HA NB-GFP/mCh-Atb2 yeast; the middle image is the dynamic change of microtubules and Mal3 within 10 min, the left and right images are blue in the middle image The enlarged view of the box is indicated by the gray dotted line; the red arrow indicates the movement of Mal3 along the microtubule to the end of the microtubule, the blue arrow indicates the disappearance of the microtubule from the microtubule Mal3 to the end of the microtubule, and the yellow arrow indicates that the microtubule remains stable; The yellow arrow indicates that the microtubule remains stable, indicating that Mal3 can maintain the stability of the microtubule. The blue arrow indicates the disappearance of Mal3, indicating that the disappearance of Mal3 may cause microtubule instability and shorten the microtubule. At the intersection of the red arrow and the blue arrow, Mal3 Back to the end of the microtubule, the microtubule begins to grow;

Figure 8 shows the scheme of connecting peptides on the surface of Nda3 protein by CRISPR technology; the left picture is the Nda3 structure in the complex structure numbered 5mjs provided by https://www.rcsb.org/; the red peptide is Y36 H37 (red arrow and red letters and numbers), the right picture is the enlarged part of the dotted box in the left picture;

Figure 9 shows the effect of protein surface labeling method applied to Nda3; the left side of the middle dotted line in Figure 9A is the image of microtubules labeled by the traditional method of fission yeast, and the right side is the image of the microtubule labelled by the new method; the right side of the middle dotted line is imaged The figure shows that the new method can label microtubules well, and compared with the traditional labeling microtubules (the left side of the dotted line), the microtubules marked by the new method are better;

Fig. 9B shows a dotted statistics diagram of the number of microtubules in each yeast cell between the microtubules labeled by the traditional method and the microtubules labeled by the new method, and the number of microtubules in the four types of yeast is basically the same;

In Figure 9C, the left and right figures are the dotted statistics of the microtubules marked by the traditional method and the microtubule lengths marked by the new method, and the percentage of the probability of microtubule length distribution. The left figure shows the GFP-Atb2 yeast microtubules The length is significantly different from the other three yeasts, and the microtubules are longer; the right figure shows that the longer microtubules of the GFP-Atb2 yeast have a higher distribution probability than the other three yeast microtubules;

Fig. 9D shows the dynamic change diagram of the microtubules marked by the traditional method and the microtubules marked by the new method within 190 seconds; it shows that the new method can label the microtubules well;

In Fig. 9E, (1)(2)(3)(4) show the growth rate, shrinkage rate, residence time of reaching the cell end, and achievable microtubules within 190 seconds between the microtubules labeled by the traditional method and the microtubules labeled by the new method. The point-like statistics of the maximum length, which are the four basic parameters of microtubule dynamics; the statistics show that the rate of microtubule shrinkage and the residence time of microtubules at the cell end of the GFP-Atb2 yeast are significantly higher than those of the other three yeast microtubules. The difference, that is, the GFP-Atb2 yeast microtubule shrinks faster and the microtubule stays at the cell end for a longer time, while the other two kinetic parameters of the four bacteria are basically the same.

Detailed ways

The invention discloses a marker or tracer composition and method for a target protein, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

(1) The N- and C-termini of Dnm1, Mal3, Nda3, etc. cannot be labeled, which will affect the protein function after labeling. To address the issue of fluorescent labeling of this protein, we have developed the novel methods described in the present invention to enable labeling of this type of protein without affecting these functions.

(2) Special design is the key point of the present invention. By referring to the protein structure information, we searched for loops that were fully exposed on the surface of the protein, and analyzed the hydrophobicity and hydrophilicity of amino acids on the loops. Then, using CRISPR-Cas9 gene editing technology, we inserted nanobodies into the loops on the surface of the protein. The identified small tag, and then use the corresponding fluorescent protein fusion nanobody (eg: nanobody-GFP) to identify and locate and track the target protein of the surface labeled nanobody to recognize the small tag (see Figure 1).

Design scheme of a new method for protein surface fluorescent labeling

In order to overcome the problem that the target protein cannot be labeled at the C-terminus or N-terminus, we have developed a new target protein labeling method. The design scheme of the new method is shown in Figure 1.

The design scheme of the new method is elaborated as follows:

(1) Look for the loop sequence facing outward on the surface of the target protein. The resolved structure of the target protein can be obtained from https://www.rcsb.org/, or based on http://www.sbg.bio.ic.ac.uk/phyre2/ and https://www.alphafold .ebi.ac.uk/ website to analyze the structure of unresolved target proteins. Next, according to the structure of the target protein, look for the loop sequence facing the surface of the target protein. These loop sequences should be avoided within the interface between the target protein and other interacting proteins.

(2) Selection of insertion sites within the loop sequence. According to the website https://dokhlab.med.psu.edu/spell/login.php, the possible multiple insertion sites of the target protein were obtained. If the predicted split insertion site is within the loop sequence identified in (1), the split insertion site is the site of interest. If the predicted split insertion site does not fall within the loop sequence identified in (1), it can be directly tried at multiple sites within the identified loop sequence and verified by subsequent experiments.

(3) To transform the target protein, insert a Nanobody identification tag with flexible amino acid linking peptides at both ends at the target site of the target protein. The CRISPR-Cas9 system can be used to connect Nanobodies with flexible amino acid linking peptides at the target site of the target protein to recognize small tags. The hydrophilic connecting peptides at both ends of the small tag can fully expose the small tag on the surface of the protein. Nanobody recognizes small tags and its two ends have flexible amino acid connecting peptides. The total number of amino acids can be controlled between 30 and 50, very small, and has good flexibility, which can largely avoid the insertion of peptides during protein folding in vivo. Folding errors caused by segment interference.

(4) Construction of Nanobody and GFP fusion expression vector. By molecular cloning method, the nanobody (Nanobody) sequence was linked with the green fluorescent protein (GFP) gene sequence in the vector to construct Nanobody-GFP plasmid.

(5) Target protein tracking. The Nanobody-GFP plasmid was transfected or transformed into cells expressing the Nanobody recognition small tag in (3), and the localization of the target protein and its dynamic localization changes were observed by fluorescence microscope.

In some preferred embodiments of the present invention, in view of the convenience of genetic manipulation of fission yeast, we choose fission yeast as the model organism and take the published HA-tagged nanobodies as representatives (different types of nanobodies and their corresponding nanobodies can be used) specific identification of small tags) to test the feasibility of the above new method. According to the above design scheme, we first used the gene editing technology based on CRISPR-Cas9 to connect two proteins within the protein sequences of Dnm1 (mitochondrial fission regulation protein), Mal3 (positive end-binding protein of microtubule) and Nda3 (β-tubulin). Dnm1, Mal3 and Nda3 proteins with surface-tagged HA were successfully traced using HANanobody-GFP with HA tags with flexible amino acid linking peptides. In addition, different nanobodies and their specific recognition small tags can also be used in this method to achieve diversified protein tracking.

Based on the above, since the traditional fluorescent labeling method is to connect fluorescent proteins at the N-terminus or C-terminus of the target protein, but some proteins are not suitable for N-terminus or C-terminus labeling, some researchers choose to use certain proteins (no specific scheme) direct insertion of fluorescent proteins. We also tried this approach where we linked GFP as well as 5xHA inside the Dnm1 protein sequence (see Figure 3A). Through microscopy imaging, we found that this method is not applicable to Dnm1, after the insertion of the two peptides in Fig. 3A into Dnm1, Dnm1 does not have normal biological function (Fig. 3B, C), which may be due to GFP, 5xHA If it is too large, it inserts inside Dnm1 and affects the spontaneous folding of Dnm1, thereby affecting Dnm1 function.

We used a small tag HA with flexible amino acid linking peptides at both ends to connect to the surface of the three proteins Dnm1, Mal3 and Nda3 (inside the protein sequence), and successfully labeled these three proteins without affecting the protein function. This peptide is GGSGGSGGSGGSGGGSGGSYPYDVPDYAGGSGGSGGSGGSGGSGGS (as shown in SEQ ID No. 1), which has only 45 amino acids, and both ends are composed of 6 groups of GGS repeating amino acids to form a flexible linking sequence. The group is very small and has a certain degree of hydrophilicity (G hydrophobic parameter is -0.4, S is -0.8), so it is easy to make the hydrophilic YPYDVPDYA(HA) protrude to the water phase during protein folding to form a hairpin structure , as long as these 45 amino acids are connected to the appropriate position of the target protein (the outer surface of the protein faces the loop), it is easy to bulge toward the water phase, so that the HA is isolated from the protein surface, thus greatly weakening the linking peptide in the protein folding process. Chain folding affects. Our experiments also justify our idea. We also demonstrated that peptides can be attached to the surface of the protein (inside the protein sequence), but it is related to the size, nature and attachment position of the attached peptides, as shown in Figure 3A,B,C.

The present invention not only provides a solution for finding the position of the connecting peptide within the protein, but also provides a small flexible connecting peptide, and provides a method for using nanobody-fluorescent protein to locate and track the surface-labeled target protein. new strategy.

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

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1 Dnm1 tracer

1.1 The N- or C-terminal fluorescent labeling of Dnm1 affects its function

In fission yeast, Dnm1 is the only protein known to directly mediate mitochondrial fission. Our study found that the mitochondrial morphology of wild-type yeast usually forms a complex network structure, and the number of mitochondria in each cell is generally multiple. When Dnm1 was deleted in fission yeast, the mitochondria did not divide, the mitochondrial morphology was abnormal and the number was 1 (see Figure 2A); the statistics of the number of mitochondria per cell is shown in Figure 2B. When the C- and N-termini of Dnm1 were marked with GFP or HA, the mitochondrial phenotype was the same as that in the absence of Dnm1 (see Figure 2A, Figure 2B). When the GFP tag was replaced with two red fluorescent protein tags, mCherry or tdTomato, the mitochondrial phenotype was also the same as that in the absence of Dnm1 (Fig. 2B). It can be seen that labeling at the C-terminus or N-terminus of Dnm1 will affect the function of Dnm1. Therefore, to trace Dnm1, a new method for fluorescent labeling of the protein surface developed by us is required.

1.2 Design of Dnm1 surface fluorescent markers and evaluation of whether surface markers affect mitochondrial morphology and dynamics

Since the labeling of both ends of Dnm1 affects protein function, we tried to label Dnm1 with a new method. First, we confirmed the outer-facing loop sequence of Dnm1 based on its predicted structure at http://www.sbg.bio.ic.ac.uk/phyre2/ ; then, based on https://dokhlab.med.psu .edu/spell/login.php website for possible multiple split insertion sites for Dnm1. Among them, two predicted cleavage insertion sites are located on the outer facing loop sequence of the protein surface, K586A587 and T680M681, respectively (see Figure 3A).

We named the target site uniformly in the way of "protein name ^TH ". If the target site is between T680 and M681, it is named as Dnm1 680 ^TH . We named the proteins connected to peptides on the outer surface of the protein toward the loop sequence in the form of "protein name-linking position ^TH connecting peptide name". For example, a HA peptide with flexible amino acid linking peptides at both ends of the connection between Dnm1 T680 and M681 (target site) is named Dnm1 680 ^TH HA.

We used CRISPR-Cas9 gene editing technology to connect GFP, 5xHA and HA to the target sites of Dnm1 586 ^TH and Dnm1 680 ^TH , respectively. Through imaging, we found that the yeast mitochondria in which the target sites of Dnm1 586 ^TH and Dnm1 680 ^TH were linked to GFP and 5xHA had abnormal mitochondrial morphology, the mitochondria did not split, and the number of mitochondria was 1 (see Figure 3B, C). However, the yeasts in which the target sites of Dnm1586 ^TH and Dnm1 680 ^TH were connected to HA had normal mitochondrial morphology and normal number of mitochondria, that is, the mitochondria could divide normally (see Figure 3B, C).

1.3 HA Nanobody-GFP localization and tracking of surface-labeled Dnm1 of HA

By molecular cloning method, the HA Nanobody (HA Nanobody) sequence was linked with the green fluorescent protein (GFP) gene sequence in the vector to construct the HA Nanobody-GFP plasmid. We transformed HA Nanobody-GFP plasmids into Dnm1 586 ^TH HA and Dnm1 680 ^TH HA strains respectively, so that HANanobody-GFP was integrated into the yeast genome, and HA Nanobody-GFP could be stably expressed in the two strains. Through microscope imaging, it was found that the mitochondrial morphology of Dnm1 680 ^TH HA/HA Nanobody-GFP was basically the same as that of wild type, and the number of mitochondria was also the same as that of wild type yeast, and Dnm1 680 ^TH HA could be recognized and localized by HANanobody-GFP (see Figure 4A, B). We imaged dnm1Δ and, Dnm1 680 ^TH HA/HA Nanobody-GFP, Dnm1-GFP, GFP-Dnm1 strains by high-resolution live cell imaging and found that Dnm1 680 ^TH HA was able to mediate mitochondrial fission (see Figure 4C) . Dnm1 has the function of splitting mitochondria. The mitochondrial oxidative phosphorylation uncoupler FCCP can promote the recruitment of Dnm1 to mitochondria. If Dnm1 function is normal, the enriched Dnm1 on mitochondria after FCCP treatment of yeast strains will cause mitochondria to be broken and fragmented. transformed form. Therefore, we treated the Dnm1680 ^TH HA/HA Nanobody-GFP strain with FCCP to further verify the function of Dnm1 680 ^TH HA. After FCCP treatment, through high-resolution live-cell imaging, we found that Dnm1 680 ^TH HA in the Dnm1 680 ^TH HA/HA Nanobody-GFP strain massively accumulated on mitochondria after a certain reaction time, and played a role in fragmenting mitochondria (see Figure 4D). It can be seen that Dnm1680 ^TH HA has a normal biological function, and our new method of fluorescent labeling on the surface of the protein can locate and observe it without affecting the function of Dnm1.

Example 2 Mal3 tracer

Mal3 is a microtubule positive end-binding protein in fission yeast and maintains microtubule stability.

2.1 The N- or C-terminal fluorescent labeling of Mal3 affects its function

We found that C- or N-terminal labeling of Mal3 resulted in very short microtubules, similar to the microtubule phenotype of Mal3-deficient cells (see Figure 5A,B). Thus, Mal3 C-terminal or N-terminal tagged GFP can affect Mal3 function. When Mal3 is out of function or functionally impaired, microtubules are unstable and prone to premature depolymerization, making it difficult to grow to wild-type microtubule lengths, thereby exhibiting a phenotype with shorter microtubules.

2.2 Design of Mal3 surface fluorescent labeling

Since the labeling of both ends of Mal3 affects protein function, we tried to label Mal3 with a new method. First, we obtained the resolved fission yeast Mal3 structure (PDB: 5m79) according to the website https://www.rcsb.org/ (see Figure 6). From this Mal3 structure, a unique off-surface facing loop sequence was found. A potential cleavage insertion site for Mal3 was then predicted according to https://dokhlab.med.psu.edu/spell/login.php, but no predicted cleavage site was found to fall within the unique surface-oriented loop sequence identified . Therefore, we analyzed the outer facing loop sequence amino acids and amino acid positions of the Mal3 surface, and chose between the most fully exposed T222 and S223 to connect the HA peptide with flexible amino acid linking peptides at both ends (see Figure 6).

2.3 Localization of HA Nanobody-GFP to track Mal3 and assess whether surface markers affect its microtubule regulatory function

We used CRISPR-Cas9 gene editing technology to insert HA peptides with flexible amino acid linking peptides at both ends between Mal3 T222 and S223. Next, the HA Nanobody-GFP plasmid was transformed into Mal3 222 ^TH HA yeast, and the Mal3 222 ^TH HA was localized and tracked using HA nanobody-GFP. Through high-resolution live-cell imaging analysis, it was found that HAnanobody-GFP could well recognize surface-tagged Mal3 with normal microtubule length (see Figure 7A,C). We compared the number of microtubules between mCherry-Atb2 yeast and Mal3 222 ^TH HA/HA nanobody-GFP yeast, and the two were basically the same (see Figure 7B). In addition, we also compared the microscopic kinetic parameters of mCherry-Atb2 yeast and Mal3 222 ^TH HA/HA nanobody-GFP yeast (see Figure 7D); the four microtubule kinetic parameters of the two yeasts were basically the same. It can be seen that the Mal3 marked by the new method does not affect the function of Mal3. We further carried out high-resolution live cell imaging observation and analysis, and found that when Mal3 disappeared from the microtubule end, the microtubule was unstable and the microtubule shortened; when Mal3 rejoined the microtubule end, the microtubule remained stable and grew (see Figure 7E). . It can be seen that our new method of protein surface fluorescent labeling can well locate and track Mal3 without affecting the biological function of Mal3.

Example 3 Nda3 localization observation

Microtubules are formed by the polymerization of alpha microtubules and beta tubulin. In Schizosaccharomyces cerevisiae, α-tubulin Nda2 and β-tubulin Nda3 are essential genes, and the two ends of the proteins are labeled and lethal. Therefore, most researchers can only label the non-essential gene, the N-terminus of α-tubulin Atb2 (the C-terminus cannot also be labeled), so as to realize microtubule observation. We used the novel method invented here to label the beta-tubulin Nda3 and assess changes in microtubule function.

3.1 Design of a new method to label β-tubulin

First, we obtained the resolved fission yeast Nda3 complex structure (PDB: 5mjs) according to the https://www.rcsb.org/ website; according to the Nda3 structure (see Figure 8), we did not interact with the target protein As the protein interaction interface, look for the loop sequence facing the surface of the target protein. Next, according to the website https://dokhlab.med.psu.edu/spell/login.php, the potential multiple insertion sites of the target protein were obtained. Among them, a cleavage site located on the outer facing loop sequence of the protein surface is Y36H37. Therefore, we chose to select HA peptides with flexible amino acid linking peptides between Y36 and H37 of Nda3 (see Figure 8).

3.2 Localization of HA Nanobody-GFP to track Nda3 and assess whether surface markers affect microtubule dynamics

We used CRISPR-Cas9 gene editing technology to connect HA peptides with flexible amino acid linking 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, respectively, and then Nda336 ^TH HA was localized by HA nanobody-GFP and HA Nanobody-yMscarlet. We successfully labeled microtubules with two colors, and the imaging effect was better than that of microtubules labeled by traditional methods (see Figure 9A). The statistical results of the number of microtubules showed that the number of microtubules in the four yeasts was basically the same (see Figure 9B). 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 9C). We further observed high-resolution live cell imaging, and the results showed that all four microtubules could shrink and grow normally (see Figure 9D). We measured the four microtubule kinetic parameters, and the results showed that the contraction rate of GFP-Atb2 microtubules was significantly different from that of the other three bacterial microtubules, the contraction rate was faster, and the GFP-Atb2 microtubules stayed at the cell end longer (see Figure 9E). mCherry-Atb2 microtubules are the most commonly used and recognized way to label microtubules in academia, and GFP-Atb2 microtubules are not completely consistent with the mCherry-Atb2 microtubule phenotype, so it can be inferred that Atb2 N-terminal There may be some problem with the labeling that affects the microtubules. The microtubule kinetic parameters marked by the new method were basically consistent with the number of mCherry-Atb2 microtubules, the length of microtubules, and the kinetics of microtubules (see Figure 9B, C, E). It can be seen that the Nda3 labelled by the new method not only does not affect its function, but can also label microtubules well, especially the green microtubules labelled by the new method, which is more effective than the traditional green labelling of microtubules.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

sequence listing

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Claims (10)

1. The application of the small label with flexible amino acid connecting peptide segment at two ends and the small label nano antibody of the fusion fluorescent protein in the target protein labeling or tracing.

2. The use of claim 1, wherein the small tag with a flexible amino acid linker at each end comprises an HA tag; the small-label nano antibody fused with the fluorescent protein comprises HA nanobody.

3. The use of claim 1 or 2, wherein the insertion position of the small tag with the flexible amino acid linker at both ends comprises any position of the loop sequence oriented outside the surface of the target protein.

4. The use of any one of claims 1 to 3 wherein the protein of interest comprises one or more of Dnm1, Mal3, Nda 3;

when the target protein is Dnm1, the insertion position is between T680 and M681; or

When the protein of interest is Mal3, the insertion position is comprised between T222 and S223; or

When the target protein is Nda3, the insertion position is between Y36 and H37.

5. The target protein labeling or tracing composition is characterized by comprising a small label with flexible amino acid connecting peptide segments at two ends and a small label nano antibody fused with fluorescent protein.

6. The labeling or labeling composition of claim 5, wherein the small tags with flexible amino acid linker peptide segments at both ends comprise HA tags; the small-label nano antibody fused with the fluorescent protein comprises HA nanobody.

7. The marking or tracing composition of claim 5 or 6, wherein the insertion position of the small tag with flexible amino acid linker peptide segments at both ends comprises any position of the loop sequence oriented outside the surface of the target protein.

8. The marking or tracing composition of any one of claims 5 to 7, wherein the target protein comprises one or more of Dnm1, Mal3, Nda 3;

when the target protein is Dnm1, the insertion position is between T680 and M681; or

When the protein of interest is Mal3, the insertion position is comprised between T222 and S223; or

When the protein of interest is Nda3, the insertion position is comprised between Y36 and H37.

9. Kit comprising a marking or tracing composition according to any of claims 5 to 8 together with an acceptable carrier or adjuvant.

10. Target protein labeling or tracing method based on a labeling or tracing composition according to any of claims 5 to 8 or a kit according to claim 9, characterized in that it comprises the following steps:

step 1: obtaining loop sequences oriented outside the surface of the target protein;

step 2: inserting small tags with flexible amino acid connecting peptide segments at two ends into the sites of the loop sequence;

and 3, step 3: adopting small-label nano antibodies of the fusion fluorescent protein corresponding to small labels with flexible amino acid connecting peptide sections at two ends to identify and position and track the target protein;

the target protein comprises one or more of Dnm1, Mal3, Nda 3;

when the target protein is Dnm1, the insertion position is between T680 and M681; or

When the protein of interest is Mal3, the insertion position is comprised between T222 and S223; or

When the target protein is Nda3, the insertion position is between Y36 and H37.

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