KR102245733B1 - Method for protein structure determination using an antibody that binds to alpha helix - Google Patents

Abstract

The present invention relates to a novel method for identifying the structure of a target protein, and more specifically, the structure of a target protein by introducing an epitope for an antihelix antibody into the alpha-helix structure of the target protein. It's about how to figure it out. With the provision of the protein identification method according to the present invention, it is possible to quickly and efficiently identify various types of protein structures that were too small in size or difficult to crystallize by conventional methods, so that the structure of the protein can be determined. It is expected to be very useful in reducing the time, cost, and effort required to do so.

Description

Method for protein structure determination using an antibody that binds to alpha helix}

The present invention relates to a method for identifying the structure of a protein, and more particularly, to a method for identifying the structure of a protein, which was difficult to determine the structure by the conventional method using an antibody that binds to an alpha helix.

Antibodies, as essential research tools in the fields of protein engineering and structural biology, have been found to be helpful in the study of the structure of proteins by X-ray crystallography and electron microscopy, and are also used to construct useful protein nanostructures. In particular, the high-resolution three-dimensional structure of a protein is an important platform in the development of a new drug because it shows the binding method of a new drug substance and a target protein. So far, about 150,000 protein structures have been identified, but among them, only 10% of all genes are human proteins that can be targeted for new drugs. Among them, in particular, the G protein-coupled receptor (GPCR), ion channel, and receptor proteins bound to the cell membrane occupy the most important positions in the development of new drugs, but their structures are hardly elucidated. The biggest reason why it is difficult to determine the structure of a protein is that it is difficult to find crystallization conditions. X-ray crystallography is the most widely used technique for protein structure identification. In order to investigate the structure using X-ray diffraction, a sample in a crystal state is required, but finding crystallization conditions is often time-consuming and impossible. Another way to determine the structure of a protein is to use a cryogenic electron microscope. This method has the advantage that it does not require a sample in a crystal state, but has a disadvantage that it is difficult to apply to small proteins with a molecular weight of 50 kDa or less. The use of antibodies is a very important tool in the identification of protein structure because it can solve these shortcomings. When an antibody binds to a target protein that is difficult to crystallize, it can help crystallization by hiding a hydrophobic or flexible surface, and it binds to a target protein of a small size, which is difficult to identify with an electron microscope, and increases the effective size of the entire protein. Make it possible.

On the other hand, antibodies useful for protein structure studies must recognize the three-dimensional conformational structure of the protein and at the same time bind to the protein in a rigid structure. Antibodies that bind to the flexible linear region of a protein cannot form a rigid complex, so even if it binds strongly to a protein, it does not help at all in the elucidation. Therefore, for each target protein, an antibody having the above-described special properties must be separately prepared. However, it takes a long time of about 6 to 8 months to produce such an antibody, and typically requires a large amount of purified target protein of about 1 to 10 mg. In other words, it takes considerable time and effort to generate antibodies and characterize complexes with antigens for the identification of the structure of proteins, especially in the case of cell membrane proteins, which are important for the development of new drugs, to produce and purify a large amount of proteins as described above. Because it is very difficult, it poses a major obstacle to the investigation of the structure.

KR 10-1426823 B1

The present invention has been devised to solve the above problems, and an object of the present invention is to provide an antihelix antibody method for clarifying the structure of a target protein.

Another object of the present invention is to provide an engineered protein of interest containing an epitope for an anti-helix antibody.

Another object of the present invention is to provide a complex in which an anti-helix antibody is bound to an engineered target protein containing an epitope for an anti-helix antibody.

Another object of the present invention is to provide a use of the engineered target protein or a complex in which an anti-helix antibody is bound to the engineered target protein to determine the structure of a target protein.

However, the technical task to be achieved by the present invention is not limited to the above-mentioned tasks, and other tasks that are not mentioned can be clearly understood by those of ordinary skill in the technical field to which the present invention belongs from the following description. will be.

In order to achieve the object of the present invention as described above, the present invention includes the step of generating an epitope for an antihelix antibody in the alpha helix of the target protein, and the amino acid sequence of the epitope Provides a method for identifying the structure of a protein of interest, characterized in that it satisfies any one or more of the following conditions (a) to (c):

(a) contains a key amino acid sequence that interacts with the anti-helix antibody;

(b) containing a hydrophobic core amino acid of the protein of interest; or

(c) When the anti-helix antibody interacts with the target protein, stereoscopic collision is not caused.

In addition, in the method for identifying the structure of a target protein according to the present invention, it provides a method for identifying the structure of a target protein, characterized in that it further comprises at least one of the following steps:

(S1) forming a complex by binding an anti-helix antibody to the target protein in which an epitope has been generated;

(S2) crystallizing the complex; And

(S3) analyzing the structure of the crystallized complex.

In one embodiment of the present invention, the core amino acid of (a) provides a method for identifying the structure of a target protein, characterized in that at least 30% of the total atomic surface area constituting the amino acid is in contact with the anti-helical antibody. .

In another embodiment of the present invention, the hydrophobic core amino acid of (b) is characterized in that at least 70% of the total atomic surface area constituting the amino acid is in contact with other atoms in the target protein. Provides a way.

In another embodiment of the present invention, the stereoscopic collision of (c) is characterized in that the distance between the anti-helix antibody and the backbone atom of the target protein is within 3.5 angstroms (Å). Provides a method for identifying structures.

In another embodiment of the present invention, the backbone atom is an alpha carbon, carboxyl group, or amino group atom of an amino acid, providing a method for identifying the structure of a target protein.

In one embodiment of the present invention, the epitope is characterized in that it is generated in the C-terminal alpha helix, the N-terminal alpha helix, or the internal alpha helix of the target protein, provides a method for identifying the structure of the target protein.

In another embodiment of the present invention, the generation of the epitope sequence is a polymerase chain reaction (PCR) using synthetic oligonucleotides, or overlap extension (OE)-PCR, or a part of a gene of a target protein comprising the epitope sequence. Or it provides a method for identifying the structure of the protein of interest, characterized in that achieved by synthesizing all.

In another embodiment of the present invention, the anti-helical antibody is represented by any one or more SEQ ID NOs selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 It provides a method for identifying the structure of a protein of interest, characterized in that it comprises an amino acid sequence.

In addition, the present invention comprises the steps of generating an epitope for an antihelix antibody in an alpha helix of a target protein; And forming a complex by binding an anti-helical antibody to the protein of interest in which the epitope is generated, wherein the amino acid sequence of the epitope satisfies any one or more of the following conditions (a) to (c). It provides a method for stabilizing the conformation of the target protein:

(a) contains key amino acids that interact with the anti-helix antibody;

(b) containing a hydrophobic core amino acid of the protein of interest; or

(c) When the anti-helix antibody interacts with the target protein, stereoscopic collision is not caused.

In addition, the present invention is an engineered protein comprising an amino acid sequence represented by any one sequence number selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 8. A (protein A), or a complex in which an anti-helix antibody is bound to the engineered protein A is provided.

In addition, the present invention provides an engineered calmodulin comprising the amino acid sequence represented by SEQ ID NO: 6, or a complex in which an anti-helix antibody is bound to the engineered calmodulin.

In addition, the present invention provides an engineered T4 lysozyme comprising the amino acid sequence represented by SEQ ID NO: 7 or a complex in which an anti-helical antibody is bound to the engineered T4 lysozyme.

In addition, the present invention provides an engineered P-gp (glycoprotein) comprising an amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 10, or a complex in which an anti-helical antibody is bound to the engineered P-gp.

In addition, the present invention is an anti-helix antibody to the engineered ABCB6 (ATP-binding cassette sub-family B member 6, mitochondrial) transporter, or the engineered ABCB6 transporter comprising the amino acid sequence represented by SEQ ID NO: 11 Provides a combined complex.

In addition, the present invention relates to the engineered protein A, the engineered calmodulin, the engineered T4 lysozyme, the engineered P-gp, the engineered ABCB6 transporter, and anti-helical antibodies to each of them. It provides the use of any one or more protein structures selected from the group consisting of bound complexes.

When the method for identifying the structure of a target protein according to the present invention is used, various types of anti-helix antibodies can be used, so that the structure of the protein is quickly and efficiently in the study of protein structure by X-ray crystallography and cryogenic electron microscopy. Can be determined, and furthermore, it is expected to be applied to construct a nanostructure of a useful protein.

1A to 1E are diagrams showing the crystal structure of the 3MNZ-Protein A #8420 complex: (FIG. 1A) the amino acid sequence of the epitope engineered in Protein A #8420; (Fig. 1b) the docking structure of protein A and gp41-3MNZ; (Fig. 1c) Predicted structure of engineered protein A with 3MNZ epitope; (Fig. 1D) Structure comparison of native protein A and engineered protein A #8420; (Fig. 2e) The crystal structure of the 3MNZ scFv and Protein A #8420 complex (left) and an enlarged view of the engineered epitope (right).
2A to 2H are diagrams showing the crystal structure of the 3LRH-Protein A complex: (FIG. 2A) the amino acid sequence of the epitope engineered in Protein A #8188; (Fig. 2b) the docking structure of protein A and huntingtin-3LRH; (Fig. 2c) Predicted structure of engineered protein A #8188 with huntingtin epitope; (Fig. 2d) The crystal structure of the 3LRH intrabody and protein A #8188 complex (left) and an enlarged view of the engineered epitope (right); (FIG. 2E) Structure comparison of native protein A and engineered protein A #8188; (FIG. 2F) Amino acid sequences of epitopes engineered in Protein A #8189 (top) and #8496 (bottom); (Fig. 2g) The crystal structure of the 3LRH intrabody and the protein A #8189 complex (left) and the crystal structure of the 3LRH intrabody and the protein A #8496 complex (right); (Fig. 2h) Structure comparison of native protein A and engineered proteins A #8189 and #8496.
3A to 3D are diagrams showing the crystal structure of the 3LRH-calmodulin complex: (Fig. 3A) the amino acid sequence of the epitope engineered in Calmodulin #9011; (Fig. 3b) structure alignment of calmodulin and huntingtin helix; (Fig. 3c) Structure comparison of native calmodulin and engineered calmodulin #9011; (Fig. 3d) The crystal structure of the 3LRH intrabody and calmodulin #9011 complex (left) and an enlarged view of the engineered epitope (right).
4A to 4D are diagrams showing the crystal structure of the 3LRH-T4 lysozyme complex: (FIG. 4A) the amino acid sequence of the epitope engineered in T4 lysozyme; (Fig. 4b) Alignment of the structure of T4 lysozyme and huntingtin helix; (Fig. 4c) The crystal structure of the 3LRH intrabody and T4 lysozyme #9213 complex (left) and an enlarged view of the engineered epitope (right); (Figure 4d) Structure comparison of native T4 lysozyme and engineered T4 lysozyme #9213.
5A to 5D are diagrams showing the crystal structure of the 1P4B-Protein A #9014 complex: (FIG. 5A) the amino acid sequence of the epitope engineered in Protein A #9014; (Fig. 5b) the predicted structure of engineered protein A with a GCN4 epitope; (Fig. 5c) The crystal structure of the 1P4B scFv and Protein A #9014 complex (left) and an enlarged view of the engineered epitope (right); (Figure 5d) Structure comparison of native protein A and engineered protein A #9014.
6A to 6F are diagrams showing the interaction between human ABCB6 and 1P4B Fab: (FIG. 6A) an overlap of the ABCB6 homology model and the NBD domain of the human ABCB6 crystal structure; (Figure 6b) the amino acid sequence of the engineered epitope in ABCB6 #4038 (top), the predicted structure of engineered ABCB6 with the 1P4B epitope (bottom left), and the predicted structure of the complex 1P4B scFv and ABCB6 #4038 (bottom right); (Fig. 6c) steric collision of 1P4B scFv and native ABCB6; (Figure 6d) size exclusion chromatography profile (left) and SDS-PAGE analysis results (right) of the ABCB6-1P4B Fab complex; (Figure 6e) Electron micrograph of ABCB6 with (left) or without (right) 1P4B Fab; (Fig. 6f) An electron microscope map showing the docking crystal structure of the bacterial complex drug ABC transporter Sav1866.
7A to 7G are diagrams showing the results of manipulating the 1P4B or 3LRH epitope in P-gp of the nematode nematode: (FIG. 7A) the predicted structure of the 1P4B scFv and P-gp #3273 complex; (Fig. 7B) The amino acid sequence of the engineered epitope at P-gp #3273 (left) and the predicted structure of the engineered P-gp with 1P4B epitope (right); (Fig. 7c) the predicted structure of the 3LRH intrabody and P-gp #3274 complex; (Fig. 7D) The amino acid sequence of the engineered epitope in P-gp #3274 (left) and the predicted structure of the engineered P-gp with 3LRH epitope (right); (Fig. 7e) Pull-down analysis of P-gp #3273-1P4B scFv complex (top) and P-gp #3274-3LRH intrabody complex (bottom) using Ni-NTA; (Fig. 7f) Size exclusion chromatography profile and SDS-PAGE analysis of P-gp #3273-1P4B scFv complex (left row) and P-gp #3274-3LRH intrabody complex (right row); (Fig. 7g) ATPase activity according to the presence or absence of an antibody stimulated by paclitaxel (left) or actinomycin D (right).

Most proteins have an alpha helix structure, and since this structure has a constant and rigid structure, antibodies that bind to it can be used to investigate the structure of the protein. The present inventors found that if the amino acid sequence of the alpha helix portion of the target protein is partially changed, the previously well-studied antibody binds strongly, and this is applied to model proteins such as protein A and calmodulin, and alpha helix. The present invention was completed by identifying the structure of the protein using model antibodies such as 3MNZ, 1P4B, and 3LRH that bind.

In one embodiment of the present invention, after generating an epitope for the 3MNZ antibody in Protein A, the crystal structure thereof was elucidated by forming a protein A-3MNZ scFv complex (see Example 2).

In another example of the present invention, after generating an epitope for 3LRH antibody on Protein A at three different positions of the alpha helix, all of them successfully interacted with 3LRH to form a stable complex (Example 3 Reference).

In another embodiment of the present invention, an epitope for the 3LRH antibody was generated on calmodulin, and it was confirmed that the calmodulin-3LRH complex did not inhibit functional and structural integrity (see Example 4).

In another example of the present invention, it was shown that a 3LRH epitope was generated on the internal alpha helix of T4 lysozyme, and a 3LRH antibody could be successfully bound thereto (see Example 5).

In another embodiment of the present invention, an epitope for the 1P4B antibody was generated in Protein A, and it was confirmed that the scFv fragments of Protein A and 1P4B form a stable complex (see Example 6).

In another embodiment of the present invention, an epitope for 1P4B antibody was generated on a human ABCB6 transporter protein whose structure is unknown, and the crystal structure of the ABCB6-1P4B Fab complex was confirmed with an electron microscope (see Example 7).

In another embodiment of the present invention, epitopes for 1P4B and 3LRH antibodies were generated in P-gp of P. nematode, and it was confirmed that these antibodies strongly interact with P-gp, and by binding of antibodies, ABC By confirming that the ATP hydrolytic activity of the transporter was inhibited, it was confirmed that the use of an anti-helix antibody can stabilize a specific conformation of the protein (see Example 8).

Through the above embodiments, the interaction between the target protein and the antibody can be induced by introducing an epitope of an anti-helix antibody, which is already known, into the alpha helix of various target proteins, which can be used to elucidate the structure of the target protein. .

Therefore, if the protein structure identification method according to the present invention is used, it is possible to quickly and efficiently determine the structure of a protein whose size is too small or crystallization is difficult, so that the structure cannot be determined by the conventional method.

The method for determining the structure of a target protein according to the present invention includes the step of generating an amino acid sequence of an epitope for an anti-helix antibody, which is well known in the past, in the alpha helix of the target protein, wherein the amino acid sequence of the epitope is an anti-helical antibody and While including the interacting core amino acids, the hydrophobic core amino acids of the target protein are preserved so that the structure of the target protein is not modified, and when the anti-helix antibody and the target protein interact, steric collision does not occur. Through this, the structure of the target protein can be efficiently and accurately identified.

In particular, by using the method for determining the structure of a target protein according to the present invention, even a small protein having a molecular weight of 50 kDa or less can be identified through an electron microscope.

As used herein, the term "epitope" is a portion of an antigen that can be recognized by a specific antibody, and an isolated or purified protein or peptide molecule that is larger than the epitope of the present invention or contains the epitope of the present invention. It is also included in the scope of the present invention.

As used herein, the terms "manipulation", "modification", or "mutation" are artificially substituted, inserted, deleted, or two or more amino acid sequences corresponding to a protein, a peptide or a portion thereof referred to herein. It is used interchangeably as meaning an action or a result of linking. In addition, the terms used in connection with an epitope, "transplantation" and "production", are substituted for one or more amino acid sequences so that part or all of the epitope sequence to which the anti-helical antibody can bind to a specific portion of the target protein may be included. , Means all actions that are deleted or added.

The term "target protein" as used herein refers to a protein whose structure is to be elucidated by the method according to the present invention, regardless of whether the structure of the protein is already known, and an alpha helical structure in the tertiary structure There is no limit to the type of protein as long as it contains.

According to one embodiment of the present invention, the "core amino acid" refers to an amino acid in which 30% or more of the total atomic surface area constituting the amino acid is in contact with the anti-helical antibody.

According to another embodiment of the present invention, the "hydrophobic core amino acid" refers to an amino acid in which 70% or more of the total atomic surface area constituting the amino acid is in contact with other atoms in the target protein.

According to another embodiment of the present invention, the "stereoscopic collision" means that the distance between the anti-helix antibody and the backbone atom of the target protein is within 3.5 angstroms (Å), thereby generating a repulsive force, and such a repulsive force is anti It acts as an element that interferes with the interaction between the helix antibody and the protein of interest. Here, the backbone atom may correspond to an alpha carbon, a carboxyl group, or an amino group atom of an amino acid constituting the target protein.

The present invention may further include, after the step of generating an epitope for an anti-helix antibody in the alpha helix of the target protein, additionally, one or more of the following steps: Anti-helical antibody to the protein of interest in which the epitope has been generated. Combining to form a complex; Or crystallizing the complex; Or analyzing the structure of the crystallized complex.

According to one embodiment of the present invention, the epitope may be generated at any one or more of the C-terminal alpha helix, the N-terminal alpha helix, or the internal alpha helix of the target protein, and the location of the epitope is of the alpha helix. According to the structure or amino acid sequence, a person of ordinary skill in the art may appropriately select it.

According to another embodiment of the present invention, the generation of the epitope sequence is a polymerase chain reaction (PCR), overlap extension (OE)-PCR using synthetic oligonucleotides, or a part of a gene of a target protein including the epitope sequence. Or may be achieved by synthesizing all, but is not limited thereto.

According to another embodiment of the present invention, the anti-helical antibody is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 It may include the amino acid sequence shown, but if an antibody capable of specifically binding to an alpha helix is not limited to the amino acid sequence listed above, preferably, the anti-helix antibody is SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; Or it may include any one of the amino acid sequence represented by SEQ ID NO: 16 and SEQ ID NO: 17.

The 3MNZ antibody used in the present invention is one of numerous antibodies produced by the gp41 peptide of HIV (human immunodeficiency virus). In this specification, to avoid confusion, the 3MNZ antibody was designated using the Protein Data Bank (PBD) ID code (see Table 4). Gp41 is a subunit of the HIV envelope protein, forming a homotrimeric complex containing six alpha helices. The 3MNZ antibody preferably comprises the amino acid sequence of SEQ ID NO: 12, and most preferably consists of the amino acid sequence represented by SEQ ID NO: 12, but is 80% or more of the amino acid sequence of SEQ ID NO: 12, more preferably 90 It may comprise an amino acid sequence having a sequence homology of% or more, even more preferably 95% or more. For example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology Includes antibodies having. The "% of sequence homology" for an amino acid sequence is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the amino acid sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (i.e., gaps) compared to not including).

The 3LRH intrabody is another example of an antihelix antibody, which is an antibody or fragment of an antibody that acts within a cell and binds to an intracellular protein. The 3LRH intrabody is an engineered variable domain fragment of an immunoglobulin light chain that binds to the 14 amino acid alpha helix section derived from human huntingtin protein. The binding site of the antigenic peptide is usually located on the concave surface formed by the beta sheet interacting with the heavy chain variable domain. When the peptide antigen binds to the 3LRH intrabody, it uses an almost ideal alpha helix structure. The 3LRH antibody preferably comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and most preferably consists of an amino acid sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14, but SEQ ID NO: 13 or SEQ ID NO: 14 It may include an amino acid sequence having at least 80%, more preferably at least 90%, even more preferably at least 95% of sequence homology with the amino acid sequence.

The 1P4B antibody recognizes helical epitopes with different amino acid sequences. It was generated and selected by ribosome display screening for the initial GCN4 peptide. The antibody binds to the 12-residue GCN4, which has an almost ideal alpha helix structure. The 1P4B antibody preferably comprises any one or more amino acid sequences of SEQ ID NO: 15, or SEQ ID NO: 16, or SEQ ID NO: 17, and most preferably is represented by SEQ ID NO: 15, or SEQ ID NO: 16, or SEQ ID NO: 17. An amino acid sequence consisting of an amino acid sequence, but having sequence homology of at least 80%, more preferably at least 90%, and even more preferably at least 95% with the amino acid sequence of SEQ ID NO: 15, or SEQ ID NO: 16, or SEQ ID NO: 17 It may include.

In addition, the present invention comprises the steps of generating an epitope for an antihelix antibody in an alpha helix of a target protein; And forming a complex by binding an anti-helical antibody to the protein of interest in which the epitope is generated, wherein the amino acid sequence of the epitope satisfies any one or more of the following conditions (a) to (c). It is possible to provide a method for stabilizing the conformation of the target protein:

(a) contains key amino acids that interact with the anti-helix antibody;

(b) containing a hydrophobic core amino acid of the protein of interest; or

(c) When the anti-helix antibody interacts with the target protein, stereoscopic collision is not caused.

In addition, the present invention is an engineered protein comprising an amino acid sequence represented by any one sequence number selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 8. A (protein A) may be provided, or a complex in which an anti-helix antibody is bound to the engineered protein A may be provided. Here, the meaning of “comprising” means that as part of the entire amino acid sequence of native protein A, the amino acid sequence of protein A engineered to generate an epitope on the alpha helical structure of native protein A may exist.

In addition, the present invention may provide an engineered calmodulin comprising the amino acid sequence represented by SEQ ID NO: 6, or a complex in which an anti-helix antibody is bound to the engineered calmodulin. Here, the meaning of "comprising" means that as part of the total amino acid sequence of native calmodulin, an amino acid sequence of calmodulin engineered to generate an epitope on the alpha helix structure of native calmodulin may exist.

In addition, the present invention may provide an engineered T4 lysozyme comprising the amino acid sequence represented by SEQ ID NO: 7 or a complex in which an anti-helix antibody is bound to the engineered T4 lysozyme. Here, the meaning of “comprising” means that as part of the entire amino acid sequence of the native T4 lysozyme, the amino acid sequence of the T4 lysozyme engineered to generate an epitope on the alpha helical structure of the native T4 lysozyme may exist.

In addition, the present invention can provide an engineered P-gp (glycoprotein) comprising an amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 10, or provide a complex in which an anti-helical antibody is bound to the engineered P-gp. have. Here, the meaning of "comprising" means that as part of the entire amino acid sequence of native P-gp, the amino acid sequence of P-gp engineered to generate an epitope on the alpha helical structure of native P-gp may exist.

In addition, the present invention provides an engineered ABCB6 (ATP-binding cassette sub-family B member 6, mitochondrial) transporter comprising the amino acid sequence represented by SEQ ID NO: 11, or against the engineered ABCB6 transporter. A complex to which a helical antibody is bound can be provided. Here, the meaning of “comprising” means that as part of the entire amino acid sequence of the native ABCB6 transporter, the amino acid sequence of the ABCB6 transporter engineered to generate an epitope on the alpha helical structure of the native ABCB6 transporter may exist.

In addition, the present invention can provide the use of the engineered protein A or the protein A structure of the complex to which the anti-helix antibody is bound thereto.

In addition, the present invention may provide a use of the engineered calmodulin or a complex to which an anti-helical antibody is bound thereto to determine the structure of calmodulin.

In addition, the present invention can provide a use of the engineered T4 lysozyme, or a complex to which an anti-helical antibody is bound thereto, to determine the structure of T4 lysozyme.

In addition, the present invention can provide the use of the engineered P-gp, or a complex to which an anti-helical antibody is bound thereto, to determine the P-gp structure.

In addition, the present invention can provide the use of the above-engineered ABCB6 transporter or a complex to which an anti-helical antibody is bound thereto to determine the structure of the ABCB6 transporter.

The terms or words used in the present specification and claims should not be construed as being limited to their usual or dictionary meanings, and the inventor may appropriately define the concept of terms in order to describe his own invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1: Experimental Materials and Methods

1.1. Production of antibodies

The antibody domain genes used in the following examples were synthesized based on the sequences deposited in the PDB protein data bank (Table 1 below).

Mutations were introduced using OE-PCR (overlap extension polymerase chain reaction). The 3LRH intrabody gene was cloned into the pET28a vector. For protein production, the protein expression plasmid was transformed into E. coli strain BL21 (DE3), and the transformed cells were transformed into LB (Lysogeny broth) medium supplemented with 50 μg/ml kanamycin at 37° C. Cultured in. When the OD 600 reached 0.7, 0.5 mM IPTG (Isopropyl β-d-1-thiogalactopyranoside) was added to induce protein production, followed by incubation at 21° C. overnight. The harvested cell pellet was resuspended in a lysis buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and homogenized with a microfluidizer (microfluidics; Microfluidic). Cell debris was removed by centrifugation, and the supernatant was loaded on a Ni-NTA (nickel-nitrilotriacetic acid) metal affinity column. The 6×histidine-tagged protein was eluted with a buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, and 500 mM imidazole (pH 8.0).

The 3MNZ scFv (single-chain variable fragment; single-chain variable fragment) gene was cloned into a pAcGP67A baculovirus transfer vector (BD Biosciences, USA). Recombinant virus was generated according to the protocol provided by the manufacturer. The 3MNZ scFv protein was expressed in High Five (BTI-Tn-5B1-4) insect cells (Invitrogen, USA) grown in SF900 medium (Invitrogen, USA). Cells infected with the recombinant baculovirus were cultured at 28° C. for 3 days. 3MNZ scFv with 6×histidine secreted in the medium was purified using Ni-NTA affinity chromatography (GE Healthcare, USA). The 1P4B scFv gene was cloned into a phagemid vector, and the transformed cells were cultured at 37° C. in CRAP medium to which 50 μg/ml of ampicillin was added. When the OD 600 reached 0.7, 0.5 mM IPTG was added to induce protein production and incubated overnight at 21°C. The harvested cells were lysed, and the antibody fragment protein was purified by Ni-NTA affinity chromatography. To remove the 6×histidine-tag, the eluted antibody fragment was incubated with thrombin overnight. For further purification of the cleaved antibody fragment, the sample was purified by injection into a Superdex 200 size-exclusion column (GE Healthcare, USA) equilibrated with 20 mM Tris (pH 8.0), 200 mM NaCl.

1.2. Preparation and pull-down analysis of engineered proteins

The mutant gene encoding the engineered epitope was generated by mutagenesis by PCR or OE-PCR using synthetic oligonucleotides (Table 2, the 4 digits after # are randomly assigned numbers for convenience of experimentation).

The gene was cloned into the expression vector pET28a, and the cloned plasmid was transformed into E. coli strain BL21 (DE3), and the transformed cells were cultured at 37°C in LB medium to which 50 μg/ml of kanamycin was added. When the OD 600 reached 0.6, 0.5 mM IPTG was added to induce protein production, and the induced cells were cultured at 37°C for an additional 4 hours. The harvested cell pellet was resuspended in lysis buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 0.5 mM PMSF, and homogenized with a microfluidizer. Cell debris was removed by centrifugation, and the supernatant was loaded onto a Ni-NTA chromatography column. 6×Histidine-attached protein was eluted with a buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, and 500 mM imidazole (pH 8.0), and already using a PD-10 desalting column (GE Healthcare, USA). The dazole was removed. To purify the calmodulin fragment, 5 mM CaCl ₂ was added to all buffers used for purification.

P-gp (glycoprotein) and human ABCB6 constructs of C. elegans encoding the engineered epitope were generated by OE-PCR using synthetic oligonucleotides, and C-terminal eGFP and 10× It was cloned into the yeast expression vector pPICZ-C with the histidine tag. The thrombin cleavage site was inserted between the protein sequence and the tag. Proteins were prepared by slightly modifying previously known methods. Briefly, the construct was transformed into Pichia pastoris strain SMD1163 (Invitrogen, USA). Cells were grown at 28° C. in MMY minimal medium to which 100 μg/mL zeocin was added, and then incubated with 1% methanol for 24 hours to induce protein production. The harvested cell pellet was frozen and crushed in liquid nitrogen by cryomilling (MM400, Retsch, Germany), 50 mM Tris (pH 8.0), 300 mM NaCl, 5 mM MgCl ₂ , 10% glycerol, 4 μg/mL DNAse. And resuspended in a lysis buffer containing 1 mM PMSF. Membrane proteins were solubilized with 2% (w/v) n-dodecyl-β-D-maltopyranoside (DDM; Anatrace, USA) for 2 hours at 4°C. Insoluble cell debris was removed by centrifugation at 90,000 g for 50 minutes, and the supernatant was loaded onto a column conjugated with Ni-NTA chromatography or anti-GFP DARPin. The C-terminal eGFP and 10×histidine tags were removed by on-column cleavage using thrombin protease. P-gp in 20 mM MES (pH 6.5), 200 mM NaCl, 5% glycerol and 0.12% (w/v) n-undecyl-β-D-maltopyranoside (n-undecyl-β-D-maltopyranoside) ; UDM; Anatrace, U.S.) eluting with a buffer containing, and ABCB6 20 mM HEPES (pH 7.5), 200 mM NaCl, 0.06% 6-cyclohexyl-1-hexyl-β-D-maltoside (6-cyclohexyl It was eluted with a buffer containing -1-hexyl-β-D-maltoside; Cymal-6; Anatrace, USA) and 0.0018% (w/v) cholesteryl hemmisuccinate. The eluted protein was concentrated with an Amicon ultra-high-speed centrifugal filter (Millipore, USA) and incubated for at least 1 hour on ice with an anti-helix antibody at a molar ratio of 1:3. Proteins with or without anti-helical antibodies were further purified by Superdex 200 gel filtration chromatography (GE Healthcare, USA).

In the pull-down analysis, eGFP with the engineered epitope and P-gp to which 10×histidine was attached were immobilized on Ni-NTA resin, and this together with purified antibody 20 mM MES (pH 6.5), 200 mM NaCl, After gently stirring for 1 hour at 4° C. in a buffer containing 5% glycerol and 0.12% (w/v) UDM, extensive washing was performed. Binding proteins were eluted from the resin in 4X Laemmli sample buffer and analyzed using 15% SDS-PAGE.

1.3. Preparation of antibody-engineered protein complex

To prepare the 3MNZ scFv-Protein A #8420 complex, the purified antibody fragment and the engineered protein were mixed at a molar ratio of 1:1 at 4° C. for 1 hour. Complex formation was observed by native-PAGE (Polyacrylamide gel electrophoresis). Other antibody-protein complexes were mixed and purified using Superdex 200 gel filtration chromatography (GE Healthcare, USA) to remove unbound protein. The antibody-protein complex was concentrated to 5 mg/mL before crystallization.

1.4. Crystallization and data collection

Crystallization conditions of the antibody-engineered protein complex and freezing conditions of the crystals are summarized in Table 3 below. All crystals were quick-frozen in liquid nitrogen at -170°C. Diffraction data was collected at the 7A and 5C beam lines of the Pohang Accelerator Research Institute (Korea). The collected diffraction data was indexed, integrated and scaled using the HKL-2000 package (HKL Research, USA).

Antibody complex Crystallization solution Cryoprotectant 3MNZ-protein A #8420 20% PEG MME 2000, 0.1 M MOPS pH 6.5, 21°C 20% ethylene glycol 3LRH-protein A #8188 35% PEG 4000, 0.1 M MOPS pH 6.5, 4°C 30% glycerol 3LRH-protein A #8189 30% PEG 4000, 0.2 M MgCl ₂ ,
0.1 M MOPS pH 6.5, 21℃ 30% ethylene glycol 3LRH-protein A #8496 35% PEG 4000, 0.1 M MES pH 5.5, 21°C 10% ethylene glycol 3LRH ^cys -protein A #8188 ^cys 45% PEG 4000, 0.1 M HEPES pH 7.5, 21°C 30% glycerol 3LRH-calmodulin #9011 50% PEG 400, 0.2 M CaCl ₂ ,
0.1 M HEPES pH 7.5, 21℃ none 3LRH-T4 lysozyme #9213 4.8 M sodium formate,
0.1 M sodium acetate pH 4.5, 21℃ 30% glycerol 1P4B-protein A #9014 1.8 M NaK phosphate,
0.1 M HEPES pH 7.5, 21℃ 30% ethylene glycol

1.5. Structure determination, purification and homology modeling

The initial phase was calculated by molecular replacement using PHASER crystallography software. The atomic model was built through iterative modeling and refinement using the COOT and PHENIX programs. Crystallographic data are shown in Table 4 below. The homology model of human ABCB6 without lysosome targeting segment (residues 1 to 205) is based on the structure of the Atm1-type ABC transporter of Novosphingobium aromaticivorans DSM 12444, NaAtm1 ( https://salilab.org/modeller). Atomic coordinates and diffraction data were deposited with the PDB.

1.6. ATP hydrolytic activity measurement

An ATP/NADH binding assay was performed to investigate the ATP hydrolytic activity of engineered P-gp in the presence and absence of antibodies. Purified proteins and drugs in surfactants were 50 mM potassium, HEPES (pH 8.0), 10 mM MgCl ₂ , 60 μg/ml pyruvate kinase, 32 μg/ml lactate dehydrogenase, 4 mM Combined in a buffer containing phosphoenolpyruvate, 0.3 mM NADH, 5 mM ATP, and 0.05% DDM, and absorbance changes at 340 nm at 25° C. for 3 minutes at 5 seconds intervals to monitor the reduction rate of NADH. It was measured. All measurements were repeated three times according to the preparation of the same protein.

1.7. Electron microscope data

In order to prepare samples for negative electron microscopy (electron microscopy, hereinafter referred to as'EM') and image acquisition, purified ABCB6 with or without 1P4B Fab was mixed with 1 mM ATP (or AMPPNP) and 1 mM MgCl _{2 on ice.} Incubated for hours. This was diluted to 0.07 mg/mL, loaded on a newly glow-discharged, carbon-coated EM grid, and stained with 0.75% (w/v) uranyl formate. I did. Data was collected on a FEI Talos L120C (FEI, Netherlands) operating at 120kV and equipped with a Ceta 16M (4k×4k) CMOS camera. The magnification was ×57,000 and the pixel size was 1.8Å.

For EM data processing and image analysis, the CTF (Contrast transfer function) parameter was estimated using the Gctf program (v1.18). A total of 390 micrographs were examined to identify approximately 150,000 particles, and extracted with a box size of 156 pixels. These were used to obtain a reference-free 2D class average using RELION-3.0. The main 2D class, in which the structural features of the ABC transporter were defined, was used to create an initial 3D model using the cryoSPARC v2 GUI interface. After the improvement of the 3D structure, the previously determined structure of Staphylococcus aureus Sav1866 was placed on a density map using a UCSF chimera.

Example 2: Generation of 3MNZ epitope of protein A and identification of crystal structure of engineered protein A-3MNZ single-chain variable fragment (scFv) complex

2.1. Generation of 3MNZ epitope in the C-terminal alpha helix of protein A

The 3MNZ antibody recognizes an alpha helical peptide having 14 amino acid residues and two gp41 C-terminal amino acids (indicated overlap in FIG. 1a). The core interaction of the peptide-antibody complex is mediated by one side of the helix consisting of the hydrophobic amino acids L61, W66 and L69 and the hydrophilic amino acids E62, D64, K65 and S68, and the other side of the helix is completely exposed to the solvent. The C-terminal N71 is not part of the helix and has a strong interaction with the heavy chain of the antibody through hydrogen bonding.

The B1 domain of protein A was selected as an experimental case for generating the 3MNZ epitope, because it consists of three alpha helices, is easily produced in E. coli , and can be crystallized under various conditions. To manipulate the protein A domain, the structure of the gp41 peptide bound to the Fab fragment of the 3MNZ antibody was superimposed on the C-terminal helix of the protein A while avoiding the steric collision of the antibody and protein A (Fig. 1b). Since three N-terminal amino acids from N56 to Q58 of gp41 do not interact with the antibody, the amino acid sequence of protein A was used for the corresponding position (Fig. 1A). In the docked structure, amino acids at positions G44, E45, K47-L49, E51, S52 and A54 of protein A play an important role in antibody binding, and thus the amino acid was changed to the gp41 amino acid sequence (Fig. Marked in red). Amino acids V42, L43, A46 and Q53 of protein A were not altered because they represent the hydrophobic core of protein A. The N50 position of protein A is not critical for binding; Therefore, alanine was selected among asparagine (Asn) or alanine (Ala) compatible with the position. #8420 Protein A produced according to the above was produced in E. coli and homogeneously purified. Protein A was judged to be structurally integrity because the protein can be overexpressed in E. coli, has great resistance to subtilisin degradation, and can be eluted as a monomer in gel filtration chromatography. . The engineered protein A was mixed with the purified scFv of the 3MNZ antibody, and the structure of the resulting complex was elucidated by X-ray crystallography.

2.2. Engineered protein A-3MNZ scFv complex crystal structure

The crystal structure of the engineered protein A-3MNZ scFv complex showed that the modification of the C-terminal helix did not substantially change the overall structure of protein A (see Figure 1e left figure), and the wild-type and engineered protein A were of 0.77Å. Cα root mean square deviation (root mean square deviation) value could be overlapped (Fig. 1d). The artificially generated epitope took an alpha helical structure as intended, and the amino acid engineered to interact with the antibody could overlap with the amino acid of gp41 bound to 3MNZ scFv (see Figure 1e right figure). Through these crystallographic observations, the present inventors demonstrated that the epitope of 3MNZ scFv can be manipulated in the C-terminal helix of Protein A.

Example 3: 3LRH epitope engineering in protein A domain

A 3LRH intrabody was selected to see if other antibodies could be used as anti-alpha helix antibodies. The antibody was chosen because the peptide-antibody interaction was mediated by one side of the helix and the other side was completely exposed to the solvent. In addition, it is easy to produce in E. coli and has a high affinity for the alpha helix antigen. The present inventors have found that three kinds of making an epitope of 3LRH at another location of said structure 3LRH and their complexes (Fig. 2a to FIG. 2 h) of protein A.

The 3LRH intrabody binds to a peptide derived from the human huntingtin protein. To create an epitope, the first 10 amino acids of the huntingtin peptide and the last 10 amino acids of the C-terminal helix of protein A were superimposed with a molecular modeling program (FIGS. 2A and 2B ). Amino acids K48, E51 and S52 of protein A were shown to be crucial for binding by targeting the antibody (Fig. 2c). Therefore, the amino acid of protein A was changed to the amino acid of huntingtin peptide. Since lysine was found at the corresponding positions in both the huntingtin peptide and protein A sequences, K48 did not need to be altered. The resulting #8188 protein A bound to 3LRH with high affinity, and the antibody-antigen complex was purified from unbound excess protein by gel filtration chromatography for crystallization. In the crystal structure, the modified C-terminal alpha helix of Protein A interacted with the antibody as intended (FIG. 2D ). The structure of the amino acid interacting with the antibody was substantially the same as that of the native huntingtin peptide 3LRH complex, and the overall structure of the mutant and native protein A overlapped with a Cα root mean square deviation value of 0.41Å as planned. (See Figure 2d right figure and Figure 2e). In addition, the introduction of a disulfide bridge can improve the stability of the 3LRH complex, which can be useful for demanding nanotechnology applications such as those occurring in animal experiments.

To demonstrate that epitopes can be generated at different locations of protein A, two additional epitope sites were created in protein A (Figure 2f; top, protein A #8189; bottom, protein A #8496). First, #8189 was created by moving the docking area by one helical turn in the docking area of #8188 Protein A. To this end, 7 amino acids of the huntingtin peptide and the C-terminal helix of protein A were overlapped to generate an artificial C-terminal epitope (top of FIG. 2f). The amino acid of the protein A structure targeting the 3LRH intrabody was changed to the amino acid of the huntingtin peptide, and the amino acid targeting the core of Protein A was not changed. The second epitope was generated by mutating the N-terminal helix of Protein A using a strategy similar to the above (Fig. 2F bottom). As intended, the two mutant proteins formed a stable complex with the 3LRH intrabody, and the complex was not dissociated by gel filtration chromatography, and their structures were confirmed. The crystal structure showed that amino acids mutated to generate the epitope interacted with the antibody as planned (FIG. 2g ), and their structures could overlap with the designed structure (FIG. 2h ).

Example 4: 3LRH epitope manipulation in Calmodulin

To investigate whether artificial helical epitopes can be produced in proteins other than protein A, the 3LRH epitope was engineered in the N-terminal helix of calmodulin (FIGS. 3A and 3B ). Calmodulin is a calcium sensor that is known to bind to calcium ions through EF-hand motifs. It is composed of N- and C-terminal domains that are structurally as well as sequence homologous. Each domain contains two calcium binding EF-hand motifs. In order to generate the 3LRH epitope, the first 8 amino acids of the calmodulin helix and the last 8 amino acids of the huntingtin helix were overlapped to dock the N-terminal helix and the huntingtin peptide of the N-terminal domain of calmodulin (Fig. 3a). As in protein A, the amino acid indicating the solvent in the overlapped region was changed to the amino acid of the huntingtin peptide (FIG. 3B). The resulting mutant N-terminal fragment of calmodulin strongly binds to the 3LRH intrabody, and the complex is able to crystallize. As designed, the structure of the calmodulin part of the calmodulin-3LRH complex did not change except for the last three amino acids, and the structure was found to be unstable in the absence of the C-terminal calmodulin fragment (Fig. 3c). . Considering that the two calcium binding sites of the engineered calmodulin fragment are occupied by calcium ions, it was confirmed that the functional and structural integrity of the protein was not impaired (refer to the left figure of Fig. 3d). The overall structure of the engineered 3LRH epitope can overlap with the structure of the huntingtin peptide in the 3LRH complex, demonstrating the success of the design (FIG. 3D ).

Example 5: manipulation of the 3LRH epitope in the inner alpha helix

In order to show that the helical epitope can be generated not only in the distal helix but also in the inner helix, a 3LRH epitope was constructed on the inner helix of T4 lysozyme. Lysozyme consists of 11 alpha helices and 3 short beta strands, and the inventors generated 3LRH epitopes in the second alpha helix (FIGS. 4A and 4B ). In order to connect the 3rd beta strand extended by 3 additional helix rotations and the 2 engineered alpha helices, a linker consisting of 12 flexible amino acids was inserted (gray dotted line in the left figure of Fig. 4c). The mutated lysozyme protein was produced in E. coli and crystallized in a complex state with a 3LRH intrabody. The crystal structure confirmed that a 3LRH epitope was generated, and that the mutated lysozyme binds to the antibody (Fig. 4c). The side chain that interacts with the antibody in the 3LRH complex has substantially the same form as that of the huntingtin peptide (see Figure 4c right figure).

Example 6: 1P4B epitope engineering in protein A domain and crystal structure of protein A-1P4B scFv complex

As the last test case for protein A, 1P4B antibody which has high affinity for the alpha helix antigen of GCN4 and can be efficiently expressed in E. coli was selected. The peptide-antibody interaction of the 1P4B antibody was again mediated by one side of the helix and the other side was completely exposed to the solvent. As in the above examples, the 1P4B-GCN4 structure was docked to the C-terminal helix of the protein A domain while avoiding steric collision between the protein A and the antibody. Subsequently, the amino acid residue interacting with the antibody was mutated to the amino acid residue of the GCN4 peptide (FIGS. 5A and 5B ). The scFv fragments of protein A and 1P4B formed a stable complex, and the purified complex was crystallized for structural analysis. The mutated protein A, #9014 binds to the antibody fragment forming the intended structure, and the amino acid mutated to bind to the antibody fragment takes substantially the same structure as that of the GCN4 peptide in the 1P4B complex (Figs. 5C and 5D). .

Example 7: Engineering the 1P4B epitope in the ABCB6 transporter

In order to confirm whether the anti-helix antibody is useful for studying the structure of an unknown membrane protein, an experiment was performed as follows. First, human ABCB6 was selected as a target protein. ABCB6 is a mitochondrial ATP binding cassette (ABC) transporter that regulates the synthesis of heme and porphyrin by translocation of coproporphyrinogen III from the cytoplasm to mitochondria. Monomer ABCB6 is a half transporter and is homogeneous, including one transmembrane domain (hereinafter referred to as'TMD') and one nucleotide binding domain (hereinafter referred to as'NBD'). It is activated by forming a dimer. The crystal structure of the isolated NBD has been revealed. However, the structure of ABCB6 including TMD has not been reported yet. Therefore, the structure was predicted using a homology modeling technique. The structure of the Atm1-type ABC exporter (NaAtm1; PDB ID code 4MRS) having 46% sequence homology with human ABCB6 was used as a template, and the program MODELER was used for calculation. The homology model of NBD was in close agreement with the crystal structure to show the accuracy of modeling (FIG. 6A), and the NBD of ABCB6 contained a short helix at the C-terminus (refer to the right picture of FIG. 6A). To transplant the 1P4B epitope, the C-terminal helix of the NBD domain of ABCB6 overlapped the last 3 amino acids of ABCB6 and the first 3 amino acids of the GCN4 helix and docked with the GCN4 peptide (FIG. 6B ). In the overlapping region, the amino acid exposed to the solvent was changed back to the amino acid of the GCN4 peptide, but the amino acid targeting ABCB6 was not changed. In addition, Q823 of ABCB6 was not in the overlapping region, but appeared to collide with the W109 and S111 residues of 1P4B scFv in the docking model (Fig. 6c). Therefore, to avoid this conflict, glutamine was changed to alanine (Q823A).

To facilitate visualization of ABCB6 in micrographs, two mutations were introduced. First, the present inventors cut the lysosomal targeting segment of human ABCB6, residues 1 to 205, in order to improve the homogeneity of the protein as shown above. And, in order to interfere with the ATP hydrolytic activity and stabilize the outward-facing structure, the E659Q mutation was introduced into the NBD domain of ABCB6. Engineered ABCB6 was produced in Pichia pastoris , and purified ABCB6 was incubated with 1P4B Fab at a molar ratio of 1:3 to form a stable complex. The complex and excess antibody obtained through this were separated using size exclusion chromatography (FIG. 6D). Subsequently, the pooled fraction containing the ABCB6-Fab complex was ^{mixed with ATP/Mg 2+} to capture ATP-bonded, outwardly catalytic ABCB6. The resulting protein complex was subjected to negative staining EM and single-particle analysis. In total, 153,945 and 138,419 particles were collected and used to obtain a two-dimensional (2D) class average of ABCB6 with or without 1P4B Fab bound, respectively. The main 2D class average of ABCB6 without bound antibody revealed the structural features of the ABC transporter: the TMD contained in the micelles of the surfactant and the NBD forming a closed dimer. In contrast, the EM image of the ABCB6-1P4B Fab complex clearly contained the extra density corresponding to the dumbbell-shaped Fab bound to the estimated NBD binding site (FIG. 6E). By 3D reconstruction, it was confirmed that the location of the Fab density coincided with the designed structure, and thus it was confirmed that the protein having the artificially designed epitope has the intended structure (FIG. 6F). Due to the flexibility between the variable and constant regions of the Fab, only the Fv region appears in the final density map.

In the structure of the present invention, only one molecule of the Fab is bound to one NBD domain because, as predicted, steric hindrance prevents binding to other domains. In the purification process, cholesteryl hemisuccinate was added to the surfactant to stabilize the structure of ABCB6 and maintain its activity, which may cause a slightly larger area of the surfactant micelle (Fig. 6f). ). Since the resolution of the EM map was not sufficient to directly build a model for ABCB6, the bacterial homologue of P-glycoprotein (hereinafter referred to as'P-gp'), and of Sav1866 of Staphylococcus aureus. The known structure was adapted to fit the final density map. As a result, it was confirmed that the EM map closely matched the Sav1866 structure, and ABCB6 showed the TMD facing outward (ie, open to the intermembrane space of the mitochondria) and the closed NBD structure.

Example 8: Inhibition of 1P4B and 3LRH epitope manipulation and ATP hydrolytic activity in P-gp

P-gp of Caenorhabditis elegans was selected as a model protein in order to test whether a specific conformation of the protein can be stabilized using an anti-helix antibody. P-gp, a member of the superfamily of ABC transporters, is a major multidrug transporter capable of imparting drug resistance by pumping anticancer drugs from cancer cells. The functional units of P-gp consist of homologous N-terminal and C-terminal halves containing TMD (TMD1 and TMD2) and cytoplasmic NBD (NBD1 and NBD2), respectively, in a single polypeptide (FIGS. 7A and 7C ). TMD recognizes a variety of substrates and facilitates translocation of the substrate across the membrane, while NBD binds to and hydrolyzes ATP, providing energy for the translocation of the substrate.

In order to introduce the 1P4B epitope into P-gp, the short helix located at the C-terminus of NBD1 was docked with the GCN4 peptide by overlapping the last 12 amino acids of the NBD1 helix with the first 12 amino acids of the GCN4 peptide (FIGS. 7A and 7B. ). The amino acid of the NBD1 helix exposed to the solvent was replaced with the amino acid of the GCN4 peptide, but the amino acid indicating the center of the NBD1 was not replaced. Using a similar strategy, a 3LRH epitope was also generated in P-gp NBD1, except that the docking region moved by one helix rotation in the docking region of 1P4B to avoid steric collision with the target protein (Figs. 7c and 7d).

A pull-down assay was performed to confirm the interaction between the engineered P-gp and the antibody in vitro. To this end, the engineered P-gp having eGFP and decahistidine tags at the C-terminus was bound to the Ni-NTA resin, and incubated with 1P4B scFv or 3LRH intrabody at a molar ratio of 1:10. After thoroughly washing, the protein mixture was separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Wild-type P-gp was used as the first negative control, and empty resin without bound P-gp was used as the second negative control to exclude the possibility of background binding. As a result, it was confirmed that the engineered P-gp strongly interacts with both antibodies (Fig. 7e). The formation of stable complexes was also confirmed by size exclusion chromatography. Both antibodies eluted in a typical high molecular weight fraction of the complex stabilized by strong interactions (Fig. 7f).

The formation of a closed NBD dimer is known to be a prerequisite for the ATPase activity of the ABC transporter. The structure-based docking model of the present invention suggested that the binding of the anti-helix antibody to the engineered P-gp sterically prevents the closing motion of the NBD domain, thereby inhibiting the ATP hydrolytic activity. To confirm this, the ATPase activity of purified P-gp in the presence and absence of antibodies was measured using paclitaxel and actinomycin D, which are potent anticancer agents, as drug substrates. As expected for the ABC transporter, all P-gp structures in the absence of antibodies showed drug-stimulated ATPase activity, and manipulation of P-gp was dependent on the drug concentration, the level of maximal stimulation by the drug, and the substrate. There was little effect on the enzymatic properties of low concentration promoting and high concentration inhibitory properties such as biphasic reaction (Fig. 7g). These results strongly suggest that implantation of the helix epitope into the target protein does not affect their intrinsic function and does not cause damage to the overall structure. However, the 1P4B scFv and 3LRH intrabody inhibited the basal ATPase activity of the engineered P-gp and the ATPase activity stimulated by the drug (FIG. 7G ). This demonstrates that the binding of the anti-helix antibody to the engineered P-gp inhibited the structural conversion of the protein to a closed state essential for ATPase activity. This was reminiscent of the effect of binding a nanobody to mouse P-gp NBD1 on ATPase activity.

Through the above embodiments, the present inventors modified amino acids exposed to the solvent of the alpha helix into a new form to interact with the antibody, so that some antibodies that bind to a specific alpha helix bind to various unrelated proteins. Showed that it can be made to do. The amino acid residues that play an important role in the structural stability of the alpha helix were not changed in order not to disturb the intrinsic structure of the protein of interest. By elucidating the structures of eight engineered protein-antibody complexes by X-ray crystallography, it was proved that the method according to the present invention is effective. In all cases, the overall structure of each mutated protein was not severely disturbed, and the mutated helix interacted with the anti-helix antibody as intended. Moreover, amino acids that directly interact with the antibody exhibited the same conformation as found in the native peptide-antibody complex.

The protein structure identification method using the anti-helix antibody according to the present invention can be used in the following situations. First, if there is structural information on a paralog or ortholog protein of a target protein, an epitope in the target protein can be designed by homology modeling using the above method. Second, it can be used when some structure of the protein complex is known. For example, if the structure of a ligand protein is known, it can be used to design an epitope in the ligand protein portion of a ligand-receptor complex whose structure is unknown. Third, the method according to the present invention may be particularly useful for structural studies of transmembrane proteins. This is because most transmembrane proteins consist entirely of long alpha helices. Antibody Fab fragments have been successfully used for structural analysis of transmembrane proteins. However, the production of high-affinity antibodies to transmembrane proteins having small hydrophilic regions requires considerable time and effort. Fourth, in drug design experiments, there is a need to determine the structure of hundreds of protein-drug complexes at high-throughput, in which the bound antibodies are crystallized or cryoelectron microscopy (cryo electron microscope) more convenient for use with high throughput. -EM) can help to identify the condition. Like many other protein engineering methods used in structural biology, the application of the anti-helix antibody approach according to the present invention requires careful control experiments to show that the functional and structural integrity of the target protein is not damaged by mutations introduced into the helix. Do.

In order to confirm the effectiveness of the method for identifying the structure of a protein according to the present invention, three antibody fragments (3MNZ, 3LRH and 1P4B) known to bind to an alpha helical peptide were used. All antibodies bound to the engineered protein to form the expected rigid structure. This success suggests that many other helix-binding antibodies can be applied to anti-helix antibody methods. Thousands of antibody-antigen structures have already been deposited with the PDB. A quick investigation into this structure revealed that more than 10 antibodies could be used as anti-helix antibodies. Moreover, since these antibodies were produced for different purposes, if a more systematic method of generating and selecting antibodies for use in the anti-helical antibody method of this study is attempted, the pool of anti-helical antibodies will rapidly increase.

In addition, in applying the anti-helix method according to the present invention, since steric collision between the antibody and the target protein interferes with mutual binding, the alpha-helix of the target protein must be well exposed to the solvent. It is believed that it would be very useful to have a panel of antibodies having different sequences and structures, which means that each antibody recognizes a different structure, and among them, antibodies that do not cause steric collisions are among the panel of candidate anti-helical antibodies. Because at least one can exist.

In conclusion, the present inventors have shown through examples that several types of previously reported antibodies that bind to the alpha helix can be used as semiuniversal anti-helix antibodies. These antibodies will be a convenient tool in the study of protein structure by X-ray crystallography and cryogenic electron microscopy. In addition, the present invention is expected to be used to assemble protein subunits into predictable structures in the field of protein nanotechnology.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Method for protein structure determination using an antibody that binds to alpha helix <130> MP19-230 <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 56 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Protein A #8420 <400> 1 Gly Ser His Met Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu Asn Met Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Leu Glu Ala 35 40 45 Asp Lys Trp Ala Ser Leu Gln Asn 50 55 <210> 2 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Protein A #8188 <400> 2 Gly Ser His Met Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu Asn Met Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala 35 40 45 Lys Lys Leu Asn Lys Ala Gln Ala Ser Leu Lys Ser Phe Gln 50 55 60 <210> 3 <211> 65 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Protein A #8189 <400> 3 Gly Ser His Met Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu Asn Met Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala 35 40 45 Lys Lys Leu Asn Lys Leu Gln Lys Ala Phe Glu Ser Leu Lys Ser Phe 50 55 60 Gln 65 <210> 4 <211> 56 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Protein A #8496 <400> 4 Met Glu Lys Leu Met Lys Ala Phe Gln Ser Leu Ala Phe Phe Glu Ile 1 5 10 15 Leu Asn Met Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala 35 40 45 Lys Lys Leu Asn Glu Ser Gln Ala 50 55 <210> 5 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Protein A #8188cys <400> 5 Gly Ser His Met Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 1 5 10 15 Leu Asn Met Pro Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln 20 25 30 Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala 35 40 45 Lys Lys Leu Asn Lys Cys Gln Ala Ser Leu Lys Ser Phe Gln 50 55 60 <210> 6 <211> 87 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Calmodulin #9011 <400> 6 Gly Ser His Met Glu Lys Leu Met Lys Ala Phe Glu Ser Leu Gln Ile 1 5 10 15 Phe Gln Phe Lys Glu Ala Phe Ser Leu Phe Asp Lys Asp Gly Asp Gly 20 25 30 Thr Ile Thr Thr Lys Glu Leu Gly Thr Val Met Arg Ser Leu Gly Gln 35 40 45 Asn Pro Thr Glu Ala Glu Leu Gln Asp Met Ile Asn Glu Val Asp Ala 50 55 60 Asp Gly Asn Gly Thr Ile Asp Phe Pro Glu Phe Leu Thr Met Met Ala 65 70 75 80 Arg Lys Met Lys Asp Thr Asp 85 <210> 7 <211> 189 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of T4 lysozyme #9213 <400> 7 Gly Ser His Met Asn Ile Phe Glu Met Leu Arg Ile Asp Glu Gly Leu 1 5 10 15 Arg Leu Lys Ile Tyr Lys Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile 20 25 30 Gly His Leu Leu Thr Lys Ser Pro Val Glu Gly Gly Gly Gly Ser Gly 35 40 45 Gly Gly Gly Ser Glu Lys Leu Met Lys Ala Phe Glu Ser Leu Ser Leu 50 55 60 Phe Gln Ala Lys Ser Glu Leu Asp Lys Ala Ile Gly Arg Asn Thr Asn 65 70 75 80 Gly Val Ile Thr Lys Asp Glu Ala Glu Lys Leu Phe Asn Gln Asp Val 85 90 95 Asp Ala Ala Val Arg Gly Ile Leu Arg Asn Ala Lys Leu Lys Pro Val 100 105 110 Tyr Asp Ser Leu Asp Ala Val Arg Arg Ala Ala Leu Ile Asn Met Val 115 120 125 Phe Gln Met Gly Glu Thr Gly Val Ala Gly Phe Thr Asn Ser Leu Arg 130 135 140 Met Leu Gln Gln Lys Arg Trp Asp Glu Ala Ala Val Asn Leu Ala Lys 145 150 155 160 Ser Arg Trp Tyr Asn Gln Thr Pro Asn Arg Ala Lys Arg Val Ile Thr 165 170 175 Thr Phe Arg Thr Gly Thr Trp Asp Ala Tyr Ala Asn Leu 180 185 <210> 8 <211> 61 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Protein A #9014 <400> 8 Gly Ser His Met Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His 1 5 10 15 Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu 20 25 30 Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Ala 35 40 45 Leu Asn His Leu Gln Asn Glu Val Ala Arg Leu Lys Lys 50 55 60 <210> 9 <211> 1325 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of P-gp #3273 <400> 9 Met Leu Arg Asn Gly Ser Leu Arg Gln Ser Leu Arg Thr Leu Asp Ser 1 5 10 15 Phe Ser Leu Ala Pro Glu Asp Val Leu Lys Thr Ala Ile Lys Thr Val 20 25 30 Glu Asp Tyr Glu Gly Asp Asn Ile Asp Ser Asn Gly Glu Ile Lys Ile 35 40 45 Thr Arg Asp Ala Lys Glu Glu Val Val Asn Lys Val Ser Ile Pro Gln 50 55 60 Leu Tyr Arg Tyr Thr Thr Thr Leu Glu Lys Leu Leu Leu Phe Ile Gly 65 70 75 80 Thr Leu Val Ala Val Ile Thr Gly Ala Gly Leu Pro Leu Met Ser Ile 85 90 95 Leu Gln Gly Lys Val Ser Gln Ala Phe Ile Asn Glu Gln Ile Val Ile 100 105 110 Asn Asn Asn Gly Ser Thr Phe Leu Pro Thr Gly Gln Asn Tyr Thr Lys 115 120 125 Thr Asp Phe Glu His Asp Val Met Asn Val Val Trp Ser Tyr Ala Ala 130 135 140 Met Thr Val Gly Met Trp Ala Ala Gly Gln Ile Thr Val Thr Cys Tyr 145 150 155 160 Leu Tyr Val Ala Glu Gln Met Asn Asn Arg Leu Arg Arg Glu Phe Val 165 170 175 Lys Ser Ile Leu Arg Gln Glu Ile Ser Trp Phe Asp Thr Asn His Ser 180 185 190 Gly Thr Leu Ala Thr Lys Leu Phe Asp Asn Leu Glu Arg Val Lys Glu 195 200 205 Gly Thr Gly Asp Lys Ile Gly Met Ala Phe Gln Tyr Leu Ser Gln Phe 210 215 220 Ile Thr Gly Phe Ile Val Ala Phe Thr His Ser Trp Gln Leu Thr Leu 225 230 235 240 Val Met Leu Ala Val Thr Pro Ile Gln Ala Leu Cys Gly Phe Ala Ile 245 250 255 Ala Lys Ser Met Ser Thr Phe Ala Ile Arg Glu Thr Leu Arg Tyr Ala 260 265 270 Lys Ala Gly Lys Val Val Glu Glu Thr Ile Ser Ser Ile Arg Thr Val 275 280 285 Val Ser Leu Asn Gly Leu Arg Tyr Glu Leu Glu Arg Tyr Ser Thr Ala 290 295 300 Val Glu Glu Ala Lys Lys Ala Gly Val Leu Lys Gly Leu Phe Leu Gly 305 310 315 320 Ile Ser Phe Gly Ala Met Gln Ala Ser Asn Phe Ile Ser Phe Ala Leu 325 330 335 Ala Phe Tyr Ile Gly Val Gly Trp Val His Asp Gly Ser Leu Asn Phe 340 345 350 Gly Asp Met Leu Thr Thr Phe Ser Ser Val Met Met Gly Ser Met Ala 355 360 365 Leu Gly Leu Ala Gly Pro Gln Leu Ala Val Leu Gly Thr Ala Gln Gly 370 375 380 Ala Ala Ser Gly Ile Tyr Glu Val Leu Asp Arg Lys Pro Val Ile Asp 385 390 395 400 Ser Ser Ser Lys Ala Gly Arg Lys Asp Met Lys Ile Lys Gly Asp Ile 405 410 415 Thr Val Glu Asn Val His Phe Thr Tyr Pro Ser Arg Pro Asp Val Pro 420 425 430 Ile Leu Arg Gly Met Asn Leu Arg Val Asn Ala Gly Gln Thr Val Ala 435 440 445 Leu Val Gly Ser Ser Gly Cys Gly Lys Ser Thr Ile Ile Ser Leu Leu 450 455 460 Leu Arg Tyr Tyr Asp Val Leu Lys Gly Lys Ile Thr Ile Asp Gly Val 465 470 475 480 Asp Val Arg Asp Ile Asn Leu Glu Phe Leu Arg Lys Asn Val Ala Val 485 490 495 Val Ser Gln Glu Pro Ala Leu Phe Asn Cys Thr Ile Glu Glu Asn Ile 500 505 510 Ser Leu Gly Lys Glu Gly Ile Thr Arg Glu Glu Met Val Ala Ala Cys 515 520 525 Lys Met Ala Asn Ala Glu Lys Phe Ile Lys Thr Leu Pro Asn Gly Tyr 530 535 540 Asn Thr Leu Val Gly Asp Arg Gly Thr Gln Leu Ser Gly Gly Gln Lys 545 550 555 560 Gln Arg Ile Ala Ile Ala Arg Ala Leu Val Arg Asn Pro Lys Ile Leu 565 570 575 Leu Leu Asp Glu Ala Thr Ser Ala Leu Asp Ala Glu Ser Glu Gly Ile 580 585 590 Val Gln Gln Ala Leu Asp Lys Ala Ala Lys Gly Arg Thr Thr Ile Ile 595 600 605 Ile Ala His Arg Leu Ser Thr Ile Arg Asn Ala Asp Leu Ile Ile Ser 610 615 620 Cys Lys Asn Gly Gln Val Val Glu Val Gly Asp His Arg Ala Leu Met 625 630 635 640 Ala Gln Gln Gly Leu Tyr Tyr Asp Leu Val Thr Ala Gln Thr Ala Thr 645 650 655 Asp His Val Asp Ser Glu Val Glu Arg Leu Lys Lys Gly Lys Phe Ser 660 665 670 Arg Glu Asn Ser Val Ala Arg Gln Thr Ser Glu His Glu Gly Leu Ser 675 680 685 Arg Gln Ala Ser Glu Met Asp Asp Ile Met Asn Arg Val Arg Ser Ser 690 695 700 Thr Ile Gly Ser Ile Thr Asn Gly Pro Val Ile Asp Glu Lys Glu Glu 705 710 715 720 Arg Ile Gly Lys Asp Ala Leu Ser Arg Leu Lys Gln Glu Leu Glu Glu 725 730 735 Asn Asn Ala Gln Lys Thr Asn Leu Phe Glu Ile Leu Tyr His Ala Arg 740 745 750 Pro His Ala Leu Ser Leu Phe Ile Gly Met Ser Thr Ala Thr Ile Gly 755 760 765 Gly Phe Ile Tyr Pro Thr Tyr Ser Val Phe Phe Thr Ser Phe Met Asn 770 775 780 Val Phe Ala Gly Asn Pro Ala Asp Phe Leu Ser Gln Gly His Phe Trp 785 790 795 800 Ala Leu Met Phe Leu Val Leu Ala Ala Ala Gln Gly Ile Cys Ser Phe 805 810 815 Leu Met Thr Phe Phe Met Gly Ile Ala Ser Glu Ser Leu Thr Arg Asp 820 825 830 Leu Arg Asn Lys Leu Phe Arg Asn Val Leu Ser Gln His Ile Gly Phe 835 840 845 Phe Asp Ser Pro Gln Asn Ala Ser Gly Lys Ile Ser Thr Arg Leu Ala 850 855 860 Thr Asp Val Pro Asn Leu Arg Thr Ala Ile Asp Phe Arg Phe Ser Thr 865 870 875 880 Val Ile Thr Thr Leu Val Ser Met Val Ala Gly Ile Gly Leu Ala Phe 885 890 895 Phe Tyr Gly Trp Gln Met Ala Leu Leu Ile Ile Ala Ile Leu Pro Ile 900 905 910 Val Ala Phe Gly Gln Tyr Leu Arg Gly Arg Arg Phe Thr Gly Lys Asn 915 920 925 Val Lys Ser Ala Ser Glu Phe Ala Asp Ser Gly Lys Ile Ala Ile Glu 930 935 940 Ala Ile Glu Asn Val Arg Thr Val Gln Ala Leu Ala Arg Glu Asp Thr 945 950 955 960 Phe Tyr Glu Asn Phe Cys Glu Lys Leu Asp Ile Pro His Lys Glu Ala 965 970 975 Ile Lys Glu Ala Phe Ile Gln Gly Leu Ser Tyr Gly Cys Ala Ser Ser 980 985 990 Val Leu Tyr Leu Leu Asn Thr Cys Ala Tyr Arg Met Gly Leu Ala Leu 995 1000 1005 Ile Ile Thr Asp Pro Pro Thr Met Gln Pro Met Arg Val Leu Arg Val 1010 1015 1020 Met Tyr Ala Ile Thr Ile Ser Thr Ser Thr Leu Gly Phe Ala Thr Ser 1025 1030 1035 1040 Tyr Phe Pro Glu Tyr Ala Lys Ala Thr Phe Ala Gly Gly Ile Ile Phe 1045 1050 1055 Gly Met Leu Arg Lys Ile Ser Lys Ile Asp Ser Leu Ser Leu Ala Gly 1060 1065 1070 Glu Lys Lys Lys Leu Tyr Gly Lys Val Ile Phe Lys Asn Val Arg Phe 1075 1080 1085 Ala Tyr Pro Glu Arg Pro Glu Ile Glu Ile Leu Lys Gly Leu Ser Phe 1090 1095 1100 Ser Val Glu Pro Gly Gln Thr Leu Ala Leu Val Gly Pro Ser Gly Cys 1105 1110 1115 1120 Gly Lys Ser Thr Val Val Ala Leu Leu Glu Arg Phe Tyr Asp Thr Leu 1125 1130 1135 Gly Gly Glu Ile Phe Ile Asp Gly Ser Glu Ile Lys Thr Leu Asn Pro 1140 1145 1150 Glu His Thr Arg Ser Gln Ile Ala Ile Val Ser Gln Glu Pro Thr Leu 1155 1160 1165 Phe Asp Cys Ser Ile Ala Glu Asn Ile Ile Tyr Gly Leu Asp Pro Ser 1170 1175 1180 Ser Val Thr Met Ala Gln Val Glu Glu Ala Ala Arg Leu Ala Asn Ile 1185 1190 1195 1200 His Asn Phe Ile Ala Glu Leu Pro Glu Gly Phe Glu Thr Arg Val Gly 1205 1210 1215 Asp Arg Gly Thr Gln Leu Ser Gly Gly Gln Lys Gln Arg Ile Ala Ile 1220 1225 1230 Ala Arg Ala Leu Val Arg Asn Pro Lys Ile Leu Leu Leu Asp Glu Ala 1235 1240 1245 Thr Ser Ala Leu Asp Thr Glu Ser Glu Lys Val Val Gln Glu Ala Leu 1250 1255 1260 Asp Arg Ala Arg Glu Gly Arg Thr Cys Ile Val Ile Ala His Arg Leu 1265 1270 1275 1280 Asn Thr Val Met Asn Ala Asp Cys Ile Ala Val Val Ser Asn Gly Thr 1285 1290 1295 Ile Ile Glu Lys Gly Thr His Thr Gln Leu Met Ser Glu Lys Gly Ala 1300 1305 1310 Tyr Tyr Lys Leu Thr Gln Lys Gln Met Thr Glu Lys Lys 1315 1320 1325 <210> 10 <211> 1325 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of P-gp #3274 <400> 10 Met Leu Arg Asn Gly Ser Leu Arg Gln Ser Leu Arg Thr Leu Asp Ser 1 5 10 15 Phe Ser Leu Ala Pro Glu Asp Val Leu Lys Thr Ala Ile Lys Thr Val 20 25 30 Glu Asp Tyr Glu Gly Asp Asn Ile Asp Ser Asn Gly Glu Ile Lys Ile 35 40 45 Thr Arg Asp Ala Lys Glu Glu Val Val Asn Lys Val Ser Ile Pro Gln 50 55 60 Leu Tyr Arg Tyr Thr Thr Thr Leu Glu Lys Leu Leu Leu Phe Ile Gly 65 70 75 80 Thr Leu Val Ala Val Ile Thr Gly Ala Gly Leu Pro Leu Met Ser Ile 85 90 95 Leu Gln Gly Lys Val Ser Gln Ala Phe Ile Asn Glu Gln Ile Val Ile 100 105 110 Asn Asn Asn Gly Ser Thr Phe Leu Pro Thr Gly Gln Asn Tyr Thr Lys 115 120 125 Thr Asp Phe Glu His Asp Val Met Asn Val Val Trp Ser Tyr Ala Ala 130 135 140 Met Thr Val Gly Met Trp Ala Ala Gly Gln Ile Thr Val Thr Cys Tyr 145 150 155 160 Leu Tyr Val Ala Glu Gln Met Asn Asn Arg Leu Arg Arg Glu Phe Val 165 170 175 Lys Ser Ile Leu Arg Gln Glu Ile Ser Trp Phe Asp Thr Asn His Ser 180 185 190 Gly Thr Leu Ala Thr Lys Leu Phe Asp Asn Leu Glu Arg Val Lys Glu 195 200 205 Gly Thr Gly Asp Lys Ile Gly Met Ala Phe Gln Tyr Leu Ser Gln Phe 210 215 220 Ile Thr Gly Phe Ile Val Ala Phe Thr His Ser Trp Gln Leu Thr Leu 225 230 235 240 Val Met Leu Ala Val Thr Pro Ile Gln Ala Leu Cys Gly Phe Ala Ile 245 250 255 Ala Lys Ser Met Ser Thr Phe Ala Ile Arg Glu Thr Leu Arg Tyr Ala 260 265 270 Lys Ala Gly Lys Val Val Glu Glu Thr Ile Ser Ser Ile Arg Thr Val 275 280 285 Val Ser Leu Asn Gly Leu Arg Tyr Glu Leu Glu Arg Tyr Ser Thr Ala 290 295 300 Val Glu Glu Ala Lys Lys Ala Gly Val Leu Lys Gly Leu Phe Leu Gly 305 310 315 320 Ile Ser Phe Gly Ala Met Gln Ala Ser Asn Phe Ile Ser Phe Ala Leu 325 330 335 Ala Phe Tyr Ile Gly Val Gly Trp Val His Asp Gly Ser Leu Asn Phe 340 345 350 Gly Asp Met Leu Thr Thr Phe Ser Ser Val Met Met Gly Ser Met Ala 355 360 365 Leu Gly Leu Ala Gly Pro Gln Leu Ala Val Leu Gly Thr Ala Gln Gly 370 375 380 Ala Ala Ser Gly Ile Tyr Glu Val Leu Asp Arg Lys Pro Val Ile Asp 385 390 395 400 Ser Ser Ser Lys Ala Gly Arg Lys Asp Met Lys Ile Lys Gly Asp Ile 405 410 415 Thr Val Glu Asn Val His Phe Thr Tyr Pro Ser Arg Pro Asp Val Pro 420 425 430 Ile Leu Arg Gly Met Asn Leu Arg Val Asn Ala Gly Gln Thr Val Ala 435 440 445 Leu Val Gly Ser Ser Gly Cys Gly Lys Ser Thr Ile Ile Ser Leu Leu 450 455 460 Leu Arg Tyr Tyr Asp Val Leu Lys Gly Lys Ile Thr Ile Asp Gly Val 465 470 475 480 Asp Val Arg Asp Ile Asn Leu Glu Phe Leu Arg Lys Asn Val Ala Val 485 490 495 Val Ser Gln Glu Pro Ala Leu Phe Asn Cys Thr Ile Glu Glu Asn Ile 500 505 510 Ser Leu Gly Lys Glu Gly Ile Thr Arg Glu Glu Met Val Ala Ala Cys 515 520 525 Lys Met Ala Asn Ala Glu Lys Phe Ile Lys Thr Leu Pro Asn Gly Tyr 530 535 540 Asn Thr Leu Val Gly Asp Arg Gly Thr Gln Leu Ser Gly Gly Gln Lys 545 550 555 560 Gln Arg Ile Ala Ile Ala Arg Ala Leu Val Arg Asn Pro Lys Ile Leu 565 570 575 Leu Leu Asp Glu Ala Thr Ser Ala Leu Asp Ala Glu Ser Glu Gly Ile 580 585 590 Val Gln Gln Ala Leu Asp Lys Ala Ala Lys Gly Arg Thr Thr Ile Ile 595 600 605 Ile Ala His Arg Leu Ser Thr Ile Arg Asn Ala Asp Leu Ile Ile Ser 610 615 620 Cys Lys Asn Gly Gln Val Val Glu Val Gly Asp His Arg Ala Leu Met 625 630 635 640 Ala Gln Gln Gly Leu Tyr Tyr Asp Leu Val Thr Ala Gln Thr Thr Lys 645 650 655 Leu Asp Lys Ala Phe Ser Ser Leu Lys Ser Phe Gln Gly Lys Phe Ser 660 665 670 Arg Glu Asn Ser Val Ala Arg Gln Thr Ser Glu His Glu Gly Leu Ser 675 680 685 Arg Gln Ala Ser Glu Met Asp Asp Ile Met Asn Arg Val Arg Ser Ser 690 695 700 Thr Ile Gly Ser Ile Thr Asn Gly Pro Val Ile Asp Glu Lys Glu Glu 705 710 715 720 Arg Ile Gly Lys Asp Ala Leu Ser Arg Leu Lys Gln Glu Leu Glu Glu 725 730 735 Asn Asn Ala Gln Lys Thr Asn Leu Phe Glu Ile Leu Tyr His Ala Arg 740 745 750 Pro His Ala Leu Ser Leu Phe Ile Gly Met Ser Thr Ala Thr Ile Gly 755 760 765 Gly Phe Ile Tyr Pro Thr Tyr Ser Val Phe Phe Thr Ser Phe Met Asn 770 775 780 Val Phe Ala Gly Asn Pro Ala Asp Phe Leu Ser Gln Gly His Phe Trp 785 790 795 800 Ala Leu Met Phe Leu Val Leu Ala Ala Ala Gln Gly Ile Cys Ser Phe 805 810 815 Leu Met Thr Phe Phe Met Gly Ile Ala Ser Glu Ser Leu Thr Arg Asp 820 825 830 Leu Arg Asn Lys Leu Phe Arg Asn Val Leu Ser Gln His Ile Gly Phe 835 840 845 Phe Asp Ser Pro Gln Asn Ala Ser Gly Lys Ile Ser Thr Arg Leu Ala 850 855 860 Thr Asp Val Pro Asn Leu Arg Thr Ala Ile Asp Phe Arg Phe Ser Thr 865 870 875 880 Val Ile Thr Thr Leu Val Ser Met Val Ala Gly Ile Gly Leu Ala Phe 885 890 895 Phe Tyr Gly Trp Gln Met Ala Leu Leu Ile Ile Ala Ile Leu Pro Ile 900 905 910 Val Ala Phe Gly Gln Tyr Leu Arg Gly Arg Arg Phe Thr Gly Lys Asn 915 920 925 Val Lys Ser Ala Ser Glu Phe Ala Asp Ser Gly Lys Ile Ala Ile Glu 930 935 940 Ala Ile Glu Asn Val Arg Thr Val Gln Ala Leu Ala Arg Glu Asp Thr 945 950 955 960 Phe Tyr Glu Asn Phe Cys Glu Lys Leu Asp Ile Pro His Lys Glu Ala 965 970 975 Ile Lys Glu Ala Phe Ile Gln Gly Leu Ser Tyr Gly Cys Ala Ser Ser 980 985 990 Val Leu Tyr Leu Leu Asn Thr Cys Ala Tyr Arg Met Gly Leu Ala Leu 995 1000 1005 Ile Ile Thr Asp Pro Pro Thr Met Gln Pro Met Arg Val Leu Arg Val 1010 1015 1020 Met Tyr Ala Ile Thr Ile Ser Thr Ser Thr Leu Gly Phe Ala Thr Ser 1025 1030 1035 1040 Tyr Phe Pro Glu Tyr Ala Lys Ala Thr Phe Ala Gly Gly Ile Ile Phe 1045 1050 1055 Gly Met Leu Arg Lys Ile Ser Lys Ile Asp Ser Leu Ser Leu Ala Gly 1060 1065 1070 Glu Lys Lys Lys Leu Tyr Gly Lys Val Ile Phe Lys Asn Val Arg Phe 1075 1080 1085 Ala Tyr Pro Glu Arg Pro Glu Ile Glu Ile Leu Lys Gly Leu Ser Phe 1090 1095 1100 Ser Val Glu Pro Gly Gln Thr Leu Ala Leu Val Gly Pro Ser Gly Cys 1105 1110 1115 1120 Gly Lys Ser Thr Val Val Ala Leu Leu Glu Arg Phe Tyr Asp Thr Leu 1125 1130 1135 Gly Gly Glu Ile Phe Ile Asp Gly Ser Glu Ile Lys Thr Leu Asn Pro 1140 1145 1150 Glu His Thr Arg Ser Gln Ile Ala Ile Val Ser Gln Glu Pro Thr Leu 1155 1160 1165 Phe Asp Cys Ser Ile Ala Glu Asn Ile Ile Tyr Gly Leu Asp Pro Ser 1170 1175 1180 Ser Val Thr Met Ala Gln Val Glu Glu Ala Ala Arg Leu Ala Asn Ile 1185 1190 1195 1200 His Asn Phe Ile Ala Glu Leu Pro Glu Gly Phe Glu Thr Arg Val Gly 1205 1210 1215 Asp Arg Gly Thr Gln Leu Ser Gly Gly Gln Lys Gln Arg Ile Ala Ile 1220 1225 1230 Ala Arg Ala Leu Val Arg Asn Pro Lys Ile Leu Leu Leu Asp Glu Ala 1235 1240 1245 Thr Ser Ala Leu Asp Thr Glu Ser Glu Lys Val Val Gln Glu Ala Leu 1250 1255 1260 Asp Arg Ala Arg Glu Gly Arg Thr Cys Ile Val Ile Ala His Arg Leu 1265 1270 1275 1280 Asn Thr Val Met Asn Ala Asp Cys Ile Ala Val Val Ser Asn Gly Thr 1285 1290 1295 Ile Ile Glu Lys Gly Thr His Thr Gln Leu Met Ser Glu Lys Gly Ala 1300 1305 1310 Tyr Tyr Lys Leu Thr Gln Lys Gln Met Thr Glu Lys Lys 1315 1320 1325 <210> 11 <211> 632 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of ABCB6 #4038 <400> 11 Met Ala Pro Gly Leu Arg Pro Gln Ser Tyr Thr Leu Gln Val His Glu 1 5 10 15 Glu Asp Gln Asp Val Glu Arg Ser Gln Val Arg Ser Ala Ala Gln Gln 20 25 30 Ser Thr Trp Arg Asp Phe Gly Arg Lys Leu Arg Leu Leu Ser Gly Tyr 35 40 45 Leu Trp Pro Arg Gly Ser Pro Ala Leu Lys Leu Val Val Leu Ile Cys 50 55 60 Leu Gly Leu Met Gly Leu Glu Arg Ala Leu Asn Val Leu Val Pro Ile 65 70 75 80 Phe Tyr Arg Asn Ile Val Asn Leu Leu Thr Glu Lys Ala Pro Trp Asn 85 90 95 Ser Leu Ala Trp Thr Val Thr Ser Tyr Val Phe Leu Lys Phe Leu Gln 100 105 110 Gly Gly Gly Thr Gly Ser Thr Gly Phe Val Ser Asn Leu Arg Thr Phe 115 120 125 Leu Trp Ile Arg Val Gln Gln Phe Thr Ser Arg Arg Val Glu Leu Leu 130 135 140 Ile Phe Ser His Leu His Glu Leu Ser Leu Arg Trp His Leu Gly Arg 145 150 155 160 Arg Thr Gly Glu Val Leu Arg Ile Ala Asp Arg Gly Thr Ser Ser Val 165 170 175 Thr Gly Leu Leu Ser Tyr Leu Val Phe Asn Val Ile Pro Thr Leu Ala 180 185 190 Asp Ile Ile Ile Gly Ile Ile Tyr Phe Ser Met Phe Phe Asn Ala Trp 195 200 205 Phe Gly Leu Ile Val Phe Leu Cys Met Ser Leu Tyr Leu Thr Leu Thr 210 215 220 Ile Val Val Thr Glu Trp Arg Thr Lys Phe Arg Arg Ala Met Asn Thr 225 230 235 240 Gln Glu Asn Ala Thr Arg Ala Arg Ala Val Asp Ser Leu Leu Asn Phe 245 250 255 Glu Thr Val Lys Tyr Tyr Asn Ala Glu Ser Tyr Glu Val Glu Arg Tyr 260 265 270 Arg Glu Ala Ile Ile Lys Tyr Gln Gly Leu Glu Trp Lys Ser Ser Ala 275 280 285 Ser Leu Val Leu Leu Asn Gln Thr Gln Asn Leu Val Ile Gly Leu Gly 290 295 300 Leu Leu Ala Gly Ser Leu Leu Cys Ala Tyr Phe Val Thr Glu Gln Lys 305 310 315 320 Leu Gln Val Gly Asp Tyr Val Leu Phe Gly Thr Tyr Ile Ile Gln Leu 325 330 335 Tyr Met Pro Leu Asn Trp Phe Gly Thr Tyr Tyr Arg Met Ile Gln Thr 340 345 350 Asn Phe Ile Asp Met Glu Asn Met Phe Asp Leu Leu Lys Glu Glu Thr 355 360 365 Glu Val Lys Asp Leu Pro Gly Ala Gly Pro Leu Arg Phe Gln Lys Gly 370 375 380 Arg Ile Glu Phe Glu Asn Val His Phe Ser Tyr Ala Asp Gly Arg Glu 385 390 395 400 Thr Leu Gln Asp Val Ser Phe Thr Val Met Pro Gly Gln Thr Leu Ala 405 410 415 Leu Val Gly Pro Ser Gly Ala Gly Lys Ser Thr Ile Leu Arg Leu Leu 420 425 430 Phe Arg Phe Tyr Asp Ile Ser Ser Gly Cys Ile Arg Ile Asp Gly Gln 435 440 445 Asp Ile Ser Gln Val Thr Gln Ala Ser Leu Arg Ser His Ile Gly Val 450 455 460 Val Pro Gln Asp Thr Val Leu Phe Asn Asp Thr Ile Ala Asp Asn Ile 465 470 475 480 Arg Tyr Gly Arg Val Thr Ala Gly Asn Asp Glu Val Glu Ala Ala Ala 485 490 495 Gln Ala Ala Gly Ile His Asp Ala Ile Met Ala Phe Pro Glu Gly Tyr 500 505 510 Arg Thr Gln Val Gly Glu Arg Gly Leu Lys Leu Ser Gly Gly Glu Lys 515 520 525 Gln Arg Val Ala Ile Ala Arg Thr Ile Leu Lys Ala Pro Gly Ile Ile 530 535 540 Leu Leu Asp Gln Ala Thr Ser Ala Leu Asp Thr Ser Asn Glu Arg Ala 545 550 555 560 Ile Gln Ala Ser Leu Ala Lys Val Cys Ala Asn Arg Thr Thr Ile Val 565 570 575 Val Ala His Arg Leu Ser Thr Val Val Asn Ala Asp Gln Ile Leu Val 580 585 590 Ile Lys Asp Gly Cys Ile Val Glu Arg Gly Arg His Glu Ala Leu Leu 595 600 605 Ser Arg Gly Gly Val Tyr Ala Asp Met Trp Ala Leu Gln His Leu Glu 610 615 620 Asn Glu Val Ala Arg Leu Lys Lys 625 630 <210> 12 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of 3MNZ scFv <400> 12 Asp Pro Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro 1 5 10 15 Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30 Asp Tyr Ser Val His Trp Val Lys Gln Val Pro Gly Lys Gly Leu Lys 35 40 45 Trp Met Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp 50 55 60 Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Ser Ser Ala Ser Thr 65 70 75 80 Ala Tyr Leu Glu Ile His Asn Leu Thr Asn Glu Asp Thr Ala Thr Tyr 85 90 95 Phe Cys Ala Leu Gly Trp Leu His Trp Gly Leu Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ala Met Ser 130 135 140 Gly Gly Gln Lys Val Thr Met Arg Cys Lys Ser Ser Gln Ser Leu Leu 145 150 155 160 Asn Ser Arg Asn Glu Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Ile Arg Glu Ser 180 185 190 Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205 Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys 210 215 220 Leu Gln His Tyr Asn Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu 225 230 235 240 Glu Ile Lys Ser Gly Arg 245 <210> 13 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of 3LRH intrabody <400> 13 Gly Ser His Met Gly Ser Gln Pro Val Leu Thr Gln Ser Pro Ser Val 1 5 10 15 Ser Ala Ala Pro Arg Gln Arg Val Thr Ile Ser Val Ser Gly Ser Asn 20 25 30 Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Ile Gln Gln Leu Pro Gly 35 40 45 Arg Ala Pro Glu Leu Leu Met Tyr Asp Asp Asp Leu Leu Ala Pro Gly 50 55 60 Val Ser Asp Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu 65 70 75 80 Thr Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Ala Ala 85 90 95 Thr Trp Asp Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys 100 105 110 Val Thr Val Leu Ser Ala 115 <210> 14 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of 3LRHcys intrabody <400> 14 Gly Ser His Met Gly Ser Gln Pro Val Leu Thr Gln Ser Pro Ser Val 1 5 10 15 Ser Ala Ala Pro Arg Gln Arg Val Thr Ile Ser Val Ser Gly Ser Asn 20 25 30 Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Ile Gln Gln Leu Pro Gly 35 40 45 Arg Ala Pro Glu Leu Leu Met Cys Asp Asp Asp Leu Leu Ala Pro Gly 50 55 60 Val Ser Asp Arg Phe Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu 65 70 75 80 Thr Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Ala Ala 85 90 95 Thr Trp Asp Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys 100 105 110 Val Thr Val Leu Ser Ala 115 <210> 15 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of 1P4B scFv <400> 15 Met Ala Asp Tyr Ala Asp Ala Val Val Thr Gln Glu Ser Ala Leu Thr 1 5 10 15 Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly 20 25 30 Ala Val Thr Thr Ser Asn Tyr Ala Ser Trp Val Gln Glu Lys Pro Asp 35 40 45 His Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly 50 55 60 Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu 65 70 75 80 Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala 85 90 95 Leu Trp Tyr Ser Asn His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr 100 105 110 Val Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Gln Gln Ser Gly Pro 130 135 140 Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser 145 150 155 160 Gly Phe Ser Leu Thr Asp Tyr Gly Val Asn Trp Val Arg Gln Ser Pro 165 170 175 Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Asp Gly Ile Thr 180 185 190 Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser Val Thr Lys Asp Asn 195 200 205 Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Ser Gly Asp 210 215 220 Ser Ala Arg Tyr Tyr Cys Val Thr Gly Leu Phe Asp Tyr Trp Gly Gln 225 230 235 240 Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Gly Ala Asp 245 250 <210> 16 <211> 211 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of 1P4B Fab Heavy chain <400> 16 Asp Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr 20 25 30 Gly Val Asn Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Ser Val Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Gly Asp Ser Ala Arg Tyr Tyr Cys Val 85 90 95 Thr Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser 100 105 110 Ser Arg Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 130 135 140 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 145 150 155 160 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 165 170 175 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr 180 185 190 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 195 200 205 His His His 210 <210> 17 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of 1P4B Fab Light chain <400> 17 Met Ala Asp Tyr Ala Asp Ala Val Val Thr Gln Glu Ser Ala Leu Thr 1 5 10 15 Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly 20 25 30 Ala Val Thr Thr Ser Asn Tyr Ala Ser Trp Val Gln Glu Lys Pro Asp 35 40 45 His Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly 50 55 60 Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu 65 70 75 80 Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala 85 90 95 Leu Trp Tyr Ser Asn His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr 100 105 110 Val Leu Arg Ser Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 115 120 125 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 130 135 140 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 145 150 155 160 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 165 170 175 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 180 185 190 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 195 200 205 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220

Claims (10)

It includes the step of generating an epitope for an antihelix antibody in the alpha helix of the target protein, and the amino acid sequence of the epitope is at least one of the conditions of the following (a) to (c). It is characterized by satisfying:
(a) contains key amino acids that interact with the anti-helix antibody;
(b) containing a hydrophobic core amino acid of the protein of interest; or
(c) no steric collision is caused when the anti-helix antibody interacts with the target protein,
In this case, the anti-helical antibody comprises an amino acid sequence represented by any one or more SEQ ID NOs selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 Characterized in that, the method for identifying the structure of the target protein.
The method of claim 1,
A method for determining the structure of a protein of interest, characterized in that it further comprises one or more of the following steps:
(S1) forming a complex by binding an anti-helix antibody to the target protein in which an epitope has been generated;
(S2) crystallizing the complex; And
(S3) analyzing the structure of the crystallized complex.
The method of claim 1,
The core amino acid of (a) is characterized in that at least 30% of the total atomic surface area constituting the amino acid is in contact with the anti-helical antibody, a method for identifying the structure of a target protein.
The method of claim 1,
In the hydrophobic core amino acid of (b), at least 70% of the total atomic surface area constituting the amino acid is in contact with other atoms in the target protein.
The method of claim 1,
The three-dimensional collision of (c) is characterized in that the distance between the anti-helical antibody and the backbone atoms of the target protein is within 3.5 angstroms (Å), the method for identifying the structure of the target protein.
The method of claim 5,
The backbone atom is an alpha carbon, carboxyl group, or amino group atom of an amino acid.
The method of claim 1,
The epitope is characterized in that it is generated in the C-terminal alpha helix, the N-terminal alpha helix, or the internal alpha helix of the target protein.
The method of claim 1,
The generation of the epitope sequence is achieved by synthesizing some or all of the genes of the target protein including the epitope sequence, PCR (polymerase chain reaction), OE (overlap extension)-PCR using synthetic oligonucleotides. Characterized in, a method for identifying the structure of a target protein.
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