KR101835014B1 - A novel Protein interference method and use of the same - Google Patents

Abstract

The present invention relates to a novel protein interference method and a use thereof, and more particularly, to a novel protein interference method and its use, and more particularly, to a novel protein interference method and a method of using the complex of ring-box protein 1 (C- The present invention relates to a method for decomposing a substrate protein in a nucleus and a cytoplasm of a cell by treating the protein with a protein and a composition used in the method. The phenotype of the cell can be confirmed by time.

Description

[0001] The present invention relates to novel protein interference methods and uses thereof,

The present invention relates to a novel protein interference method and its use.

Human cells are composed of different proteins. Numerous proteins are enzymes and hormones that cause chemical reactions. They regulate the function of the cells by selectively degrading the proteins in the cells. Therefore, there is a protein ubiquitination system as a mechanism that causes a specific protein to be degraded.

In addition to these functions, protein ubiquitination is a post-translational modification of DNA repair, mRNA export, regulation of cell cycle, protein interactions in various signal transduction mechanisms, protein trafficking, and ubiquitinated proteins to form aggresome to cause autophagy. It is involved in various functions.

Protein ubiquitination is caused by ubiquitin. Ubiquitin is a regulatory protein consisting of 76 amino acids. Subsequent actions of three enzymes, E1, E2, and E3, lead to ubiquitination, which binds to the substrate protein. E1 catalyzes the acyl-adenylation of ubiquitin C terminus by binding to ubiquitin under conditions with ATP as an active enzyme. Ubiquitin bound to E1 moves to E2, which is a conjugating enzyme, to form a complex and binds to E3, a ligase enzyme. And the amine of the lysine group in the substrate forms a covalent bond with the glycine and nucleophilic attack at the C terminal of ubiquitin, so ubiquitin migrates from E2 to substrate protein.

This protein mechanism occurs repeatedly and ubiquitin composed of 7 lysines binds to another ubiquitin to form a polyubiquitin chain. There are seven kinds of Ubiquitin chains, and they are divided into K6, K11, K27, K29, K33, K48, and K63 depending on which residue is bonded. And functions in a variety of ways depending on the ubiquitin chain bound to the protein (Figure 1A).

Ubiquitin - Proteasome  System (UPS)

Typically, the degradation of proteins in cells is mediated by lysosomes and proteasomes. There is a major difference between these two mechanisms: lysosomes do not have selectivity or regulation ability, whereas proteasome has selectivity or control ability.

Proteasome is present in the nucleus and cytoplasm and degrades most of the proteins bound to the ubiquitin chain. Proteasome consists of two subunits, 19S regulatory particle and 20S core particle. The 19S regulatory particle recognizes proteins bound to the ubiquitin chain and induces degradation of the protein into peptides by 20S core particles. (Figure 1A)

E3 ligase

E3 ligase binds both E2 and substrate proteins and has the specificity to determine substrate proteins degraded by proteasome through ubiquitination. E3 ligase is classified into two types according to HECT (Homologous to E6-AP carboxyl terminus) domain and RING (Really Interesting New Gene) -finger domain (Pintard L, Willems A, Peter M. EMBO J. 2004; 8): 1681-7.).

Unlike the HECT E3 ligase, which contains cysteine, which acts as an intermediate for transferring ubiquitin to the substrate protein, the RING-finger E3 ligase directly transfers ubiquitin to the substrate protein without an intermediate mediator. The RING finger domain binds directly to E2 and has ligase activity. In addition, cysteine and histidine are structurally safe by 'cross-brace' generated by binding zinc ions.

RING-finger E3 ligase functions through the interaction of proteins. It is composed of Skp1, Cullin1, F-box protein and is composed of SCF complex and Rbx1, Cul2, Elongin B, Elongin C and SOCS- The -CBC complex is well known. Proteins with the BTB domain bind to each other to form dimers, which bind to Cul3 at the C-terminus and function as E3 ubiquitin ligases (Deshaies RJ, Joazeiro CA. Annu Rev Biochem. 2009; 78: 399-434).

One) Rbx1

Rbx1 (RING-box protein 1) is a protein that constitutes the SCF complex known as CRL (cullin-based RING ligase) and binds to E2 and cullin to function as an E3 ligase.

Rbx1 has a RING motif that binds to the zinc ions to stabilize the structure. Two large loops and H3, which is an a-helix structure, bind to E2 and play a role in maintaining the structure. H3 exists at the C-terminus and plays an important role in Rbx1 function. In addition, cullin binds to the N-terminus. 47 (3): 371-7. (1999). In this study, we have investigated the effect of a single- 82.) (Fig. 1 (B))

2) SPOP

SPOP (Speckle-type POZ protein) is a substrate-specific adapter consisting of a BTB domain in which a MATH domain and a cullin3 (Cul3) bind to a substrate protein. In the BTB domain, there is a dimer-forming moiety, which binds proteins with the BTB domain and binds ubiquitin bound to E2 to the substrate protein. The MATH domain determines the specificity of the substrate protein and regulates the amount of protein present in the cell. A variety of substrate proteins of SPOP commonly have a motif that binds SPOP (Zhuang M, Calabrese MF, Liu J, et al., Mol Cell. 2009; 36 (1): 39-50). Recent studies have shown that MATH domain changes cause prostate cancer (Gan W et al. 2015). The BTB domain contains a dimer domain (DD) that causes the dimer structure of SPOP and a 3-box that binds to the N terminus of the subset Cul3 (Zhuang M et al. 2009). (Figure 1C)

Genome editing is a type of genetic engineering that manipulates the genotype of an organism. Genetic editing involves double-stranded breaks of the gene of interest through nucleic acid degrading enzymes and by homologous recombination or non-homologous end joining by the cell's own machinery Manipulate the gene. This method is used to knock-out the desired gene and study the function of the protein.

Genetic editing uses nucleic acid degrading enzymes and mainly meganucleases, Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and CRISPR / Cas systems. Mega nucleic acid degrading enzymes have high specificity due to their long recognition sequences, but their cost and time are inefficient to target all sequences in the genome. On the other hand, ZFNs and TALENs can be used in all nucleotide sequences without limitation, but they have low specificity and thus are toxic to cells. The CRISPR / Cas system is highly specific for Cas9 protein that cuts DNA and gRNA which recognizes specific DNA sequence. The most commonly used RNA interference (RNAi) method is easy to experiment because it manipulates genes on mRNA by using sequence-specific inhibition of gene expression by dsRNA called short interfering RNA (siRNA).

However, the gene editing method that is used until now has the function of off target effect which cuts DNA base sequence similar to the desired DNA base sequence and the case that the cell does not change its phenotype through the adaptation period, It is unsuitable for use as a method of study.

Recently, Ab-SPOP has been used to overcome this problem and to rapidly break down the proteins in the nucleus (Shin YJ, Park SK, Jung YJ, et al., Sci.

However, this technique can not be used to study proteins present in the cytoplasm, because it acts only in the nucleus. It may also disrupt observation in a short time.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned problems, and it is an object of the present invention to provide a method of detecting a protein existing entirely in the nucleus and cytoplasm of a cell through nanobody and knocking down the cell under specific conditions (Protein-i) technology that can be used in the future.

In order to accomplish the above object, the present invention provides a method of treating a substrate protein with a complex of a nano-body binding to a substrate protein and a ring-box protein 1 (C-terminally removed) Thereby providing a method for degrading the substrate protein.

In one embodiment of the present invention, the C-terminal deleted RING-box protein 1 is preferably composed of the amino acid sequence of SEQ ID NO: 1, but it is preferable that the R- All mutants which achieve the effects of the present invention through mutation are also included within the scope of the present invention.

In another embodiment of the present invention, the substrate protein is preferably a protein existing in a cell nucleus and / or a cytosol, and the substrate protein is preferably a fluorescent protein, but is not limited thereto.

In one embodiment of the present invention, the nanobody binds to a protein existing in a cell nucleus or cytosol, but is not limited thereto.

In a preferred embodiment of the present invention, the complex of the nano-body and the C-terminal removed ring-box protein 1 (RING-box protein 1) is preferably overexpressed but not limited thereto.

The present invention also provides a composition for degrading a substrate protein comprising a complex of a nano-body binding to a substrate protein and a ring-box protein 1 (C-terminal removed) as an active ingredient.

Hereinafter, the present invention will be described.

In the present invention, a protein interference (Protein-i) technology capable of recognizing proteins existing throughout the nucleus and cytoplasm of a cell through a nanobody using the E3 ubiquitin ligase and knocking down the cell under specific conditions to observe the phenotype of the cell was developed .

The term "nanobody" of the present invention is generally defined in International Patent Application Publication No. WO08 / 020079 or WO < RTI ID = 0.0 > 09 / 138,519 & (E. G., Optimized for chemical stability and / or solubility, maximum overlap with known human framework regions and maximal expression). It will be appreciated that the term nanobodies or nanobodies may be referred to as Nanobodies < RTI ID = 0.0 > and / or < / RTI > nanobodies ' because they are registered trade names of Ablynx.

The newly developed protein interference (Protein-i) of the present invention was synthesized on E3 ubiquitin ligase using an antibody that binds to a desired protein. Therefore, by using the antibody of the desired protein, it is labeled by ubiquitination under specific conditions (conditions with doxycycline) and degraded by the proteasome. This technique works faster than existing knock-down methods and does not perform genetic manipulation, so it solves problems caused by nonspecific cleavage and cell adaptation. Unlike the developed nanobody-SPOP, it can decompose the proteins present in the cell precursors, and because it acts slowly, the expression level of the cells that can be seen by decreasing the protein level can be confirmed with time.

Figure 1 shows the principle of protein ubiquitination. Ubiquitination is a three-step process; Ubiquitin activation, conjugation and ligation are performed by E1, E2, and E3. As a result of this process, ubiquitin binds to the substrate, and this process is repeated several times to make the ubiquitin chain, which causes proteolysis through the proteasome.
Figure 2 is a model of Cullin-RING E3 ligases. (A) Rbx1 binds to E2-ubiquitin and substrate (F-box protein, VHL-box, BTB protein) complexes. (B) SPOP consists of BTB and MATH domains. The BTB domain binds to the Cullin3-Rbx1-ubiquin complex and the MATH domain binds to the substrate.
Figure 3 shows FACS analysis of GFP / 293tetOn stable cell lines transiently transfected with various synthetic E3 ligase complexes, Rbx1. After transfection, each candidate and RFP670 is expressed by doxycycline treatment from the bidirectional TRE promoter. Doxycycline was added for FACS analysis for 36 h. Transfection-free GFP / 293tetOn stable cell line. Cells RFP670, vhhGFP4-SNoFbox, vhhGFP4- NSlim, vhhGFP4-Rbx1, vhhGFP4 -Rbx1 delN, vhhGFP4 -Rbx1 delC, vhhGFP4 -Rbx1 ^delNdelC , vhhGFP4mutation -Rbx1 expressing ^delC .
Figure 4: Microscopic analysis of GFP / 293tetOn stable cell lines transiently transfected with various synthetic E3 ligase complexes, Rbx1. After transfection, each Candidate and TagRFP was expressed from a bidirectional TRE promoter by doxycycline treatment. Doxycycline was added for 24 hours for microscopic analysis. Each panel represents a combination of GFP (green), TagRFP (red), and GFP and TagRFP (yellow).
Figure 5: Microscopic analysis of GFP / 293tetOn stable cell line transiently transfected with a synthetic cullin complex. Nanobody -Cul1 ¹ And Nanobody -Cul3 ¹ deletes all Rbx1 binding domains. Each panel represents the combination of Hoechst (blue) GFP (green), TagRFP (red), and GFP and TagRFP (yellow). Hoechst is a blue fluorescent dye used for DNA staining.
Figure 6 shows FACS analysis of several GFP fusion protein / 293tetOn stable cell lines transiently ^transfected with the nano-body-synthetic E3 ubiquitin ligase complex, Ab-Rbx1 ^delC .
Figure 7: Microscopic analysis of cMycGFP, TAFGFP, CBRGFP, and p27GFP / 293tetOn stable cell lines transiently ^transfected with the nano-body-synthetic E3 ubiquitin ligase complex, Ab-Rbx1 ^delC and Ab-SPOP.
Figure 8 is a microscopic analysis of GFP / 293tetOn stable cell lines transiently ^transfected with several ^Nanobody -synthetic E3 ubiquitin ligase complexes, Ab-Rbx1 ^delC .
Figure 9 shows that three mCherry ^nanobodies fused to Rbx1 ^delC also form an active synthetic E3 ligase. 293TetOn cells stably expressing mCherry are transfected as a vector expressing Ab-Rbx1 ^delC . GFP expression and mCherry defects were measured 36 h after addition of doxycycline (1 μg / ml) to the medium. Selective defects of mCherry were observed only in cells ^transfected with Ab-Rbx1 ^delC . LaM2, LaM3, and LaM4 are mCherry nanobodies.
Figure 10 depicts the nanobody-synthetic E3 ubiquitin ligase complex, Ab-Rbx1 ^delC And Ab _mutation -Rbx1 ^delC . Microscopy and FACS analysis of the inserted stable cell line. (A) Microscopic analysis. Three candidates for Ab-Rbx1 ^delC . Doxycycline is added for 24 hours for microscopic analysis. Each panel displays the combination of GFP (green), TagRFP (red), and GFP and TagRFP (yellow). (B) Analysis of GFP by FACS

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as being limited by the following examples.

Example  1. Strain and culture conditions

1) Escherichia coli strain and culture

In the present invention, Escherichia coli Top10F '(F' [lacIq Tn10 (tetR)] mcrA (mrr-hsdRMS-mcrBC) f80lacZM15 lacX74 deoR nupG recA1 araD139 (ara-leu) 7697 galU galK rpsL StrR), Invitrogen) was used. Most of the strains were cultured in LB medium (1% Tryptone, 0.5% yeast extract, 0.5% sodium chloride, Lennox) for 12 to 15 hours at 37 ℃. Amplicillin (100 ug / ml) and kanamycin (25 ug / ml) were added to LB medium depending on the type of plasmid.

2) Animal cells and culture

293TetOn cells were cultured in DMEM (with 4.5 g / L glucose, L-glutamine, sodium pyruvate) supplemented with 10% FBS, 1% penicillin / streptomycin and 1% HEPES at 5% CO 2 and 37 ° C. To maintain GFP fluorescence in the cells, 293TetOn-CMV-GFP cells were cultured in DMEM (supplemented with 4.5 g / L glucose, L-glutamine, sodium pyruvate [10% FBS, % penicillin / streptomycin, 1% HEPES]) medium (5% CO 2, 37 ° C). L-glutamine, sodium pyruvate, [10% FBS, 1 [mu] g / ml) was added to 293TetOn-CMV-GFP cells using pEM791_SRa_HygroB plasmid in the presence of Hygromycin % penicillin / streptomycin, 1% HEPES]) medium (5% CO 2, 37 ° C).

Example  2. Vector construction

1) Expression vector construction of animal cells

The fluorescent protein gene and the E3 ubiquitin ligase were recombined with the tetracycline response element (TRE) promoter, which initiates bi-directional expression on the pEM791-SRa-HygroB_rtTA3 plasmid. TRE promoter was used to express only in the presence of doxycycline.

Therefore, it was selectively used according to the method of analyzing the fluorescent protein. First, the vector required for the analysis through a microscope was amplified by PCR with the fluorescent gene TagRFP in the TRE promoter and the restriction enzyme (BstB / Sal) was treated (Table 1). The vector required for the analysis by FACS was purchased from MitoRFP670 (Addgene: 45462), amplified by PCR, and then subjected to restriction enzyme (BstB / Sal) for recombination (Table 2) .

VhhGFP4 (deGraFP) was synthesized by attaching Flag to various E3 ubiquitin ligases with Rbx1 ^deletion (Rbx1 ^delC ), N terminus elimination (Rbx1 ^delN ), and removal of both C and N terminus (Rbx1 ^delNdelC ) (FLAG-vhhGFP4_Rbx1 ^delN FLAG-vhhGFP4_Rbx1 ^delNdelC FLAG-vhhGFP4_Rbx1 and FLAG-vhhGFP4_Rbx1 ^delC SEQ ID NOS: 2 to 5, respectively). The vector used for the negative control was vhhGFP4 by removing 3 CDRs to make a vhhGFP4 _mutation and then recombining by restriction enzyme (BsiW / Xho) treatment.

GFP fusion proteins (CBRGFP, AIMP2GFP, CD63GFP, CD64GFP, CDK4GFP, cMycGFP, DX2GFP, ELF3GFP, hPin1GFP, p27GFP, and TAF9GFP) in which GFP obtained by PCR and specific proteins were synthesized were transformed with restriction enzymes (AIMP2GFP, CBRGFP, CD63GFP, CD64GFP , CDK4GFP, cMycGFP, DX2GFP, ELF3GFP, hPIN1GFP, P27GFP, and TAF9GFP sequences are shown in SEQ ID NOS: 6 to 16, respectively).

Finally, the LaG and LaM nanobodies classified according to the affinity of the nanobody were recombined by restriction enzyme (BsiW / Xho) treatment (Table 3).

vector characteristic   ^- pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_MitoRFP670_UBCPro_TRE__globinUTR _LoxP vhhGFP4-RbxI pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_MitoRFP670_UBCPro_TRE_FLAG_vhhGFP4_RbxI_stop__globinUTR _LoxP vhhGFP4-RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_MitoRFP670_UBCPro_TRE_FLAG_vhhGFP4_RbxI_delC_stop__globinUTR _LoxP vhhGFP4-RbxI ^delN pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_MitoRFP670_UBCPro_TRE_FLAG_vhhGFP4_RbxI_delN_stop__globinUTR _LoxP vhhGFP4-RbxI ^{delN _ delC} pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_MitoRFP670_UBCPro_TRE_FLAG_vhhGFP4_RbxI_delN_delC_stop__globinUTR _LoxP vhhGFP4 _mut- RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_MitoRFP670_UBCPro_TRE_FLAG_vhhGFP4mutation_RbxI_delN_delC_stop__globinUTR _LoxP vhHGFP4-NSlim pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_MitoRFP670_UBCPro_TRE_FLAG_vhhGFP4_NSlim_stop__globinUTR _LoxP vhhGFP4-SNoFbox pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_MitoRFP670_UBCPro_TRE_FLAG_vhhGFP4_SNoFbox_stop__globinUTR _LoxP

Table 1 shows the FACS analysis vector

Vector Description vhhGFP4-RbxI pSRa_ HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_vhhGFP4_RbxI_stop__globinUTR _LoxP vhhGFP4-RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_vhhGFP4_RbxI_delC_stop__globinUTR _LoxP vhhGFP4-RbxI ^delN pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_vhhGFP4_RbxI_delN_stop__globinUTR _LoxP vhhGFP4-RbxI ^{delN _ delC} pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_vhhGFP4_RbxI_delN_delC_stop__globinUTR _LoxP vhhGFP4 _mut- RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_vhhGFP4mutation_RbxI_delN_delC_stop__globinUTR _LoxP vhhGFP4-SPOP pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_vhhGFP4_SPOP_stop__globinUTR _LoxP vhhGFP4 _mut- SPOP pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_vhhGFP4mutation_SPOP_stop__globinUTR _LoxP vhhGFP4 -Cul1 ^delNdelC1 pEM791_SRa_Hygro_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_vhhGFP4_Cul1delNdelC_deletion of Rbx1 binding site vhhGFP4 -Cul3 ^delNdelC1 pEM791_SRa_Hygro_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_vhhGFP4_Cul3delNdelC_partial deletion of Rbx1 binding site vhhGFP4-Cul1 ^delNdelC1- Rbx1 ^delN pEM791_SRa_Hygro_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPr ev_UBCPro_TRE_FLAG_vhhGFP4_Cul1delNdelC_deletion of Rbx1 binding site_Rbx1_delN vhhGFP4-Cul3 ^delNdelC1- Rbx1 ^delN pEM791_SRa_Hygro_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_vhhGFP4_Cul3delNdelC_partial deletion of Rbx1 binding site_Rbx1_delN vhhGFP4-Cul1 ^delNdelC- Rbx1 ^delN pEM791_SRa_Hygro_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_vhhGFP4_Cul1delNdelC_Rbx1_delN vhhGFP4-Cul3 ^delNdelC- Rbx1 ^delN pEM791_SRa_Hygro_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_vhhGFP4_Cul3delNdelC_Rbx1_delN

Table 2 shows the vector for microscopic analysis

Vector Description ^GBP1 -RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_GBP1_RbxI_delC_stop__globinUTR _LoxP ^GBP2 -RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_GBP2_RbxI_delC_stop__globinUTR _LoxP GBP6-RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_GBP6_RbxI_delC_stop__globinUTR _LoxP GBP7-RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_GBP7_RbxI_delC_stop__globinUTR _LoxP LaG2-RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_LaG2_RbxI_delC_stop__globinUTR _LoxP LaG9-RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_LaG9_RbxI_delC_stop__globinUTR _LoxP LaG14-RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_LaG14_RbxI_delC_stop__globinUTR _LoxP LaG16-RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_LaG16_RbxI_delC_stop__globinUTR _LoxP LaG18-RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_LaG18_RbxI_delC_stop__globinUTR _LoxP LaG41-RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_LaG41_RbxI_delC_stop__globinUTR _LoxP LaM2-RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_LaM2_RbxI_delC_stop__globinUTR _LoxP LaM3-RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_LaM3_RbxI_delC_stop__globinUTR _LoxP LaM4-RbxI ^delC pSRa_HygroB_IRES_rtTA3_bGHUTR_SV40UTR_TagRFPrev_UBCPro_TRE_FLAG_LaM4_RbxI_delC_stop__globinUTR _LoxP

Table 3 shows Rbx1 ^delC for various nanobody affinity vector

Example  3. Transformation of recombinant vector

1) Transformation of E. coli

During the cloning of DNA, the vector and the inserted gene were ligated into competent cells Escherichia coli Top10F 'and left on ice for 5 minutes. Heat shock was applied at 42 ° C to allow DNA to enter into competent cells. After left on ice for 3 minutes, LB medium was added and incubated at 37 ° C to give a period of recovery of the cells. Finally, LB agar medium containing necessary antibiotics was cultured for about 14 to 16 hours.

2) Transformation of animal cells

The cells were cultured on a 100p plate and treated with 0.25% trypsin (1.5 ml). Cells were collected and inoculated at a concentration of cells / well in a 6-well plate. After 36 hours, a mixture of 2 ug of DNA, 250 μl of Opti-MEM, and 8 ug of polyethylenimine (PEI) was inoculated for 15 min at room temperature. After incubation at 37 ° C for 4 hours, the medium was removed and 2 ml of DMEM medium supplemented with 2 ug doxycycline was added to each well.

Example  4. Fluorescence Analysis

1) Fluorescence analysis using fluorescence microscope

The fluorescence microscope used in the present invention was observed with GFP and TagRFP using Olympus IX71 (inverted microscope) in which pE-2 LED was immobilized. Images viewed through a microscope were analyzed with an Andor Clara CCD camera and analyzed using Metamorph (Molecular devices) software. For fluorescence analysis, the slide glass was covered with a cover glass and analyzed with a PBS. The fluorescence analysis was carried out by dropping the yeast on a slide glass with PBS and covering it with a cover glass.

2) Fluorescence analysis using flow cytometer

(1) Cell treatment for flow cytometer analysis

Cells were harvested by treatment with 0.25% trypsin in each well. The recovered cells were diluted by adding 100 ul of 500 ul to a BD Falcon FACS tube containing 1 ml of 1 × PBS (Phosphate-buffered saline).

(2) Flow cytometer analysis

FACS analysis was performed using GFP (Excitation: / Emission: 509) and RFP670 (Excitation: 643 / Emission: 670) using the FACScaliber ^TM System. GFP was excited by using a 488nm laser and emission was confirmed by FL1 (530/30 bandpass filter) emission. RFP670 uses a 635nm laser to induce an excitation state and identify the emitted emission with the FL4 (661/16 bandpass filter). Data were analyzed using Kaluza (Backman coulter) analysis program.

Example  5. Western blot

1) Cell lysate

Plate media was removed and 1.5 ml of ice-cold PBS was added to the plate, which was then untied and transferred to a 1.5 ml microfuge tube. The supernatant was removed by centrifugation (3000 rpm, 5 min, 4 ° C) and the cell pellet was resuspended in 1.5 ml of ice-cold PBS. After removing the PBS through centrifugation (3000 rpm, 1 min, 4 ° C), the cell pellet was suspended in 600 μl of lysis buffer and transferred to a 500 μl tube. The cell lysate was incubated in a cold-room tube rotator (capable of end-over-end inversion / speed = 11) for 30 min and the supernatant was obtained via centrifugation (12000 rpm, 15 min, 4 ° C). 50% GammaBind G sepharose bead slurry (50% GammaBind G sepharose: 10ul Lysis buffer + 10ul GammaBind G sepharose) was added to the thus obtained sample and allowed to stand for 1 hour in a cold-room tube rotator. After brief centrifugation (12,000 rpm, 4 ° C), samples were obtained except beads.

2) Immunoprecipitation

The antibody was diluted in PBS, centrifuged (12,000 rpm, 4 ° C), and placed in the prepared cell lysate with 10% BSA. Incubate in a cold-room tube rotator for 6-10 hours and add 50% GammaBind G sepharose bead slurry washed with PBS.

Then, the supernatant was removed by incubation in a cold-room tube rotator for 1 hour and centrifugation (12,000 rpm, 4 ° C). The sample was sufficiently washed with PBS to prepare a sample.

3) Transfer, blocking, and development

Sample was added to the sample buffer (1X SDS-PAGE loading buffer, 100 mM DTT) to denature (95 ° C, 5 minutes). After spin-down, the sample was subjected to SDS-PAGE gel and SDS-PAGE electrophoresis (120V, 90 min). The PVDF membrane was immersed in methanol for 5 min and then transferred to transfer buffer (25 mM Tris-Base / 192 mM Glycine / 0.0375% SDS / 20% Methanol) for more than 10 min. The sample in SDS-PAGE gel was transferred (100V, 1 hr) to the PVDF membrane and the membrane was blocked with 15 ml of blocking buffer (5% milk in TBST) for 1 h. The cells were washed with 10 ml of TBST (50 mM pH 8.0 Tris-HCl, 140 mM NaCl, 0.05% Tween-20) to allow complete removal of the milk. The membrane was then incubated overnight in a cold room with a primary antibody (in TBST containing 2% BSA (Sigma, A2153-100G)) and the membrane was washed with TBST. The secondary antibody with HRP was then incubated at room temperature for 1 hour and washed with TBST. ECL detection reagents (ECL: Peroxide = 1: 1) were sprayed onto the membrane to sensitize HRP to fluorescence.

The results of the above embodiment are as follows.

Ab - Rbx1 ^delC ligase Acts as a whole cell.

Cullin-RING E3 ubiquitin ligase, Rbx1 binds to E2-ubiquitin complex and binds to Cullin and acts as a bridge. Unlike SPOP, Rbx1 does not directly bind to substrate proteins, so it synthesizes nanobodies so that they can bind directly to substrate proteins. Nanobody is an antibody that has only a variable domain that recognizes an antigen. It has a much lower molecular weight than a general antibody. Therefore, it was used in the experiment because it did not give much influence to the cells.

Since GFP is used as a substrate protein in the experiment, we synthesized vhhGFP4, a GFP nanobody at the N terminus of Rbx1, and examined how it functions in 293tetOn cells expressing GFP. Rbx1 (vhhGFP4-Rbx ^delN) removing the N-terminal, C-terminal to remove Rbx1 (vhhGFP4- Rbx ^delC), N-terminal and C-terminal to remove ^{Rbx1 (vhhGFP4-Rbx delNdelC),} vhhGFP4-Rbx1 not have the ability to recognize GFP ( vhhGFP4mut-Rbx1) and other E3 ubiquitin ligases (vhhGFP4-N-Slim, vhhGFP4-SNoFbox) were compared and analyzed. SNoFbox is an NSlim that removes the F-box domain that the adapter protein binds in the Skp1-cullin1-F-box E3 ubiquitin ligase complex. In other words, it was used as a negative control of NSlim.

First, to measure the intensity of GFP fluorescence, the recombinant Rbx1 and RFP670 were simultaneously expressed in FACS using a vector. The x-axis of the FACS data represents the intensity of GFP, and the y-axis represents RFP670. The transfected cells expressed RFP670, so we observed how the GFP intensity changed when RFP670 was expressed. As a result, it was confirmed that only vhhGFP4-Rbx ^delNdelC weakened or almost no GFP intensity (Fig. 3).

In order to visually identify GFP fluorescence through a glow-light microscope, the recombinant Rbx1 and TagRFP were simultaneously analyzed using a fluorescence microscope. As a result, it was observed that vhhGFP4-Rbx delC ^cleaves GFP from both the nucleus and the cytoplasm of the cells in the same manner as the FACS results (Fig. 4).

Nanobody-synthesized SPOPs were known to act only in nuclei (Shin YJ et al. 2015), whereas Ab-Rbx1 ^delC , which can act both on the nucleus and cytoplasm of the cells, was found.

Ab - Rbx1 ^delC ligase Do not act directly.

In previous experiments, the ^nanobody -Rbx1 delC ^acted on the cell ^precursor , where Rbx1 ^delC extends the C-terminus and affects H3, loop 2, and S4. The C-terminus is known to be the part of E2 binding. However, it was confirmed that Rbx1 with the N terminus removed by cullin did not function properly. Therefore, we used cullin to test whether the nanobody-Rbx1 ligase affects the whole cell by any change.

Cullin can be classified as 1, 2, 3, 4A, 4B, 5, 7, 9, of which Cul1 and Cul3 are related to Rbx1. Cul1 is a protein that constitutes the SCF (Skp1-Cul1-F_Box protein) complex. The C-terminus binds to Rbx1 and the N-terminus binds to the adapter Skp1. Cul3 is a protein that constitutes the CRL (Cullin-RING ubiquirin ligase) complex and binds to a protein with the BTB domain by its N-terminal. Therefore, fusion protein synthesized with Cul1 / Cul3 and Rbx1 was recombinantly used. In order to perform experiments, we synthesized nanobody (vhhGFP4-Cul1 ^delNdelC1, vhhGFP4 -Cul3 ^delNdelC1 ) in Cul1 and Cul3 which mutated N-terminal, C-terminal and Rbx1-binding sites so that Rbx1 and adapters existing in the cell do not bind. Cul1 ^delNdel11 and Cul1 ^delNdel11 synthesized in this way were ^substituted with Rbx1 (vhhGFP4-Cul1 ^delNdel11- Rbx1 ^delN , vhhGFP4-Cul3 ^delNdelC1 -Rbx1 ^delN ). In addition, Cul1 ^delNdel1 with N-terminal and C-terminal removed and ^Rbx1 with N-terminal removed from Cul3 delNdel1 (vhhGFP4-Cul1 ^delNdelC- Rbx1 ^delN , vhhGFP4-Cul3 ^delNdelC -Rbx1 ^delN ). These recombinant vectors were transfected into GFP-expressing cells to determine what changes were made.

As a result, vhhGFP4-Cul1 ^delNdelC1 and In vhhGFP4-Cul1 delNdelC11, ^Rbx1 , which is present in the cell, can not bind with adapters. Theoretically, ubiquitination does not occur. However, fluorescence microscopy analysis shows that GFP is lost or aggregated in the nucleus. This phenomenon may result from vhhGFP binding to GFP and not by degradation in the nucleus but by migration to the cytoplasm. Also, the results were not changed even in the presence of Rbx1 with N-terminal removed.

However, Cul3 ^delNdelC synthesized with Rbx1 ^without N terminus In the case of Cul1 ^delNdelC , the GFP present in the nucleus and cytoplasm of some cells was disappeared. Since Rbx1 present in the cell binds to Cul3 and is linked to E2-ubiquitin and may be degraded by UPS, GFP appears to disappear. These results suggest that Rbx1, which E2 does not bind to, binds to cullin, binds to other proteins, and is linked to E2 to function as an E3 ligase (FIG. 5).

Ab - Rbx1 ^delC ligase Various GFP  dissolve the fusion protein.

In the previous experiment, experiments were conducted with cells expressing only GFP. We then examined the ability of the fusion protein, which synthesizes other proteins in GFP, to be removed by the nanobody, vhhGFP4. Thus, cells expressing the fusion proteins CBRGFP, AIMP2GFP, CD63GFP, CD64GFP, CDK4GFP, cMycGFP, DX2GFP, ELFGFP, hPin1GFP mucin / p27GFP and TAFGFP were transfected and the GPF fluorescence intensity was measured by FACS. I checked.

FACS analysis revealed that the membrane-expressing CD63 and CD64, AIMP2, and DX2 expressed in the cells were not affected by ^nanobody -Rbx1 ^delC . However, the remaining fusion proteins showed weak GFP intensity (Fig. 6), and microscopic observation of poorly ablated CBR, AIMP2, cMyc, p27, and TAF. Comparing ^nanobody -Rbx1 ^delC with nanobody-SPOP, which degrades GFP only in nuclei, we found that, unlike nanobody-SPOP, nanobody-Rbx1 ^delC eliminated GFP in nucleus and cytoplasm. (Fig. 7)

Nanobody - Rbx1 ^delC ligase Is influenced by affinity.

In the previous experiment, it was confirmed that vhhGFP4-Rbx1 delC ^degrades GFP and fusion proteins synthesized with various proteins. Like vhhGFP4, there are many nanobodies that bind to GFP but differ in their affinity to GFP (Table 4). We determined the effect of Rbx1 on the degradation of GFP according to the affinity of Nanobody.

The nanobody used in the experiments was recombined into Rbx1 delC instead of vhhGFP4 with ^GBP1 / GBP2 / GBP6 / GBP7 / LaG2 / LaG9 / LaG14 / LaG16 / LaG18 / LaG42 (Fridy PC et al, 2014).

GBP is a GFP binding protein that binds well with GFPDP, with GBP1 having the highest affinity. It has an affinity similar to that of vhhGFP4. GFP aggregation was observed, but most of the cells were found to be degraded. Also LaG14. It was ^also observed that Rbx1 ^delC synthesized with LaG16 ^also degranely ^transfected GFP. But GBP2, GBP7, LaG18, and a Rbx1 ^delC synthesized LaG42 it was confirmed that no action. LaG18 and LaG42 have poor affinity among synthesized nanobodies. However, it was confirmed that Rbx1 ^delC synthesizing GBP6, LaG2, and LaG9 specifically ^excluded GFP existing in the nucleus or did not have GFP in the nucleus as it migrated to the cytoplasm (FIG. 8).

Nanobody - Rbx1 ^delC ligase silver nanobody To selectively degrade the protein.

So far, the experiment has been carried out using a nanobody that binds to GFP. To confirm that the ^nanobody binding to other substrate proteins ^could be synthesized in Rbx1 ^delC and that other substrate proteins other than GFP could be disappeared, we observed that mCherry, a fluorescent protein, was degraded using LaM, a nanobody that combines with mCherry. Rbx1 synthesized in ^delC The LaM nanobody is LaM2, LaM3, and LaM4, with different affinities (Table 4). In the cells expressing mCherry, it was confirmed that LaM3 and Lam4 slowly degraded mCherry, and LaM2 slowly degraded mCherry but aggregation of mCherry occurred in some cells (Fig. 9).

Nanobody MV (Da) K _d (nM) LaG2 15,919 19 ^a , 16 LaG9 16,062 3.5 LaG14 16,002 1.9 LaG16 16,306 0.7 LaG18 16,459 3,800 ^a LaG42 15,490 600 LaM2 15,151 0.49 LaM3 15,196 1.9 LaM4 14,866 0.18

Table 4 shows the properties of GFP and mCherry-specific nanobodies

LaG is a GFP nanobody and LaM is a mCherry nanobody. kd values are quantitative measurements of nanobody affinity for GFP and mCherry binding.

Nanobody - Rbx1 ^delC ligase Is affected by the level of expression.

Microscopy revealed that ^nanobody -Rbx1 ^delC degrades or aggregates fluorescent proteins such as GFP and mCherry. Protein aggregation refers to the phenomenon of aggregation of proteins because the function of the proteasome present in the cells does not occur properly. In many cases where protein aggregation occurs, the misfolded protein produced by the endoplasmic reticulum of the cell is degraded by UPS (ubiquitin-proteasome system), where the activity of the proteasome is not sufficient and the unfractionated misfolded protein binds to each other There is a case of bundling. (Hao R et al., 2013). Therefore, in order to confirm that aggregation occurs due to the aggregation of undegraded GFP due to insufficient activity of proteasome, which is limited in the cell, compared to ^nanobody -Rbx1 ^delC overexpressed by the TRE promoter, ^nanobody -Rbx1 ^delC was used with hygromycin And inserted into the cell genome to decrease the expression level. As a result, ^nanobody -Rbx1 ^delC When expressed, it was confirmed that GFP was completely degraded without aggregation in the cells (Fig. 10A).

As a result of measurement of the GFP intensity by FACS, it was found that the ^nanobody -Rbx1 ^delC was decreased by more than 10 times as much as the GFP intensity of the original cell (Fig. 10B)

<110> KNU-Industry Cooperation Foundation <120> A novel protein interference method and use of the same <130> HY160028 <160> 16 <170> Kopatentin 2.0 <210> 1 <211> 85 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 1 Met Asp Val Asp Thr Pro Ser Gly Thr Asn Ser Gly Ala Gly Lys Lys   1 5 10 15 Arg Phe Glu Val Lys Lys Trp Asn Ala Val Ala Leu Trp Ala Trp Asp              20 25 30 Ile Val Val Asp Asn Cys Ala Ile Cys Arg Asn His Ile Met Asp Leu          35 40 45 Cys Ile Glu Cys Gln Ala Asn Gln Ala Ser Ala Thr Ser Glu Glu Cys      50 55 60 Thr Val Ala Trp Gly Val Cys Asn His Ala Phe His Phe His Cys Ile  65 70 75 80 Ser Arg Trp Leu Lys                  85 <210> 2 <211> 199 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 2 Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Thr Met Asp Gln Val   1 5 10 15 Gln Leu Val Glu Ser Gly Gly Gly Ser Leu              20 25 30 Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Val Asn Arg Tyr Ser Met          35 40 45 Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val Ala Gly      50 55 60 Met Ser Ser Ala Gly Asp Arg Ser Ser Tyr Glu Asp Ser Val Lys Gly  65 70 75 80 Arg Phe Thr Ile Ser Arg Asp Asp Ala Arg Asn Thr Val Tyr Leu Gln                  85 90 95 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val             100 105 110 Asn Val Gly Phe Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser         115 120 125 Ser Leu Glu Asn Cys Ala Ile Cys Arg Asn His Ile Met Asp Leu Cys     130 135 140 Ile Glu Cys Gln Ala Asn Gln Ala Ser Ala Thr Ser Glu Glu Cys Thr 145 150 155 160 Val Ala Trp Gly Val Cys Asn His Ala Phe His Phe His Cys Ile Ser                 165 170 175 Arg Trp Leu Lys Thr Arg Gln Val Cys Pro Leu Asp Asn Arg Glu Trp             180 185 190 Glu Phe Gln Lys Tyr Gly His         195 <210> 3 <211> 180 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 3 Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Thr Met Asp Gln Val   1 5 10 15 Gln Leu Val Glu Ser Gly Gly Gly Ser Leu              20 25 30 Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Val Asn Arg Tyr Ser Met          35 40 45 Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val Ala Gly      50 55 60 Met Ser Ser Ala Gly Asp Arg Ser Ser Tyr Glu Asp Ser Val Lys Gly  65 70 75 80 Arg Phe Thr Ile Ser Arg Asp Asp Ala Arg Asn Thr Val Tyr Leu Gln                  85 90 95 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val             100 105 110 Asn Val Gly Phe Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser         115 120 125 Ser Leu Glu Asn Cys Ala Ile Cys Arg Asn His Ile Met Asp Leu Cys     130 135 140 Ile Glu Cys Gln Ala Asn Gln Ala Ser Ala Thr Ser Glu Glu Cys Thr 145 150 155 160 Val Ala Trp Gly Val Cys Asn His Ala Phe His Phe His Cys Ile Ser                 165 170 175 Arg Trp Leu Lys             180 <210> 4 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 4 Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Thr Met Asp Gln Val   1 5 10 15 Gln Leu Val Glu Ser Gly Gly Gly Ser Leu              20 25 30 Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Val Asn Arg Tyr Ser Met          35 40 45 Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val Ala Gly      50 55 60 Met Ser Ser Ala Gly Asp Arg Ser Ser Tyr Glu Asp Ser Val Lys Gly  65 70 75 80 Arg Phe Thr Ile Ser Arg Asp Asp Ala Arg Asn Thr Val Tyr Leu Gln                  85 90 95 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val             100 105 110 Asn Val Gly Phe Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser         115 120 125 Ser Leu Glu Met Asp Val Asp Thr Pro Ser Gly Thr Asn Ser Gly Ala     130 135 140 Gly Lys Lys Arg Phe Glu Val Lys Lys Trp Asn Ala Val Ala Leu Trp 145 150 155 160 Ala Trp Asp Ile Val Val Asp Asn Cys Ala Ile Cys Arg Asn His Ile                 165 170 175 Met Asp Leu Cys Ile Glu Cys Gln Ala Asn Gln Ala Ser Ala Thr Ser             180 185 190 Glu Glu Cys Thr Val Ala Trp Gly Val Cys Asn His Ala Phe His Phe         195 200 205 His Cys Ile Ser Arg Trp Leu Lys Thr Arg Gln Val Cys Pro Leu Asp     210 215 220 Asn Arg Glu Trp Glu Phe Gln Lys Tyr Gly His 225 230 235 <210> 5 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 5 Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Thr Met Asp Gln Val   1 5 10 15 Gln Leu Val Glu Ser Gly Gly Gly Ser Leu              20 25 30 Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Val Asn Arg Tyr Ser Met          35 40 45 Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val Ala Gly      50 55 60 Met Ser Ser Ala Gly Asp Arg Ser Ser Tyr Glu Asp Ser Val Lys Gly  65 70 75 80 Arg Phe Thr Ile Ser Arg Asp Asp Ala Arg Asn Thr Val Tyr Leu Gln                  85 90 95 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val             100 105 110 Asn Val Gly Phe Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser         115 120 125 Ser Leu Glu Met Asp Val Asp Thr Pro Ser Gly Thr Asn Ser Gly Ala     130 135 140 Gly Lys Lys Arg Phe Glu Val Lys Lys Trp Asn Ala Val Ala Leu Trp 145 150 155 160 Ala Trp Asp Ile Val Val Asp Asn Cys Ala Ile Cys Arg Asn His Ile                 165 170 175 Met Asp Leu Cys Ile Glu Cys Gln Ala Asn Gln Ala Ser Ala Thr Ser             180 185 190 Glu Glu Cys Thr Val Ala Trp Gly Val Cys Asn His Ala Phe His Phe         195 200 205 His Cys Ile Ser Arg Trp Leu Lys     210 215 <210> 6 <211> 1680 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 6 atgccgatgt accaggtaaa gccctatcac gggggcggcg cgcctctccg tgtggagctt 60 cccacctgca tgtaccggct ccccaacgtg cacggcagga gctacggccc agcgccgggc 120 gctggccacg tgcaggaaga gtctaacctg tctctgcaag ctcttgagtc ccgccaagat 180 gatattttaa aacgtctgta tgagttgaaa gctgcagttg atggcctctc caagatgatt 240 caaacaccag atgcagactt ggatgtaacc aacataatcc aagcggatga gcccacgact 300 ttaaccacca atgcgctgga cttgaattca gtgcttggga aggattacgg ggcgctgaaa 360 gacatcgtga tcaacgcaaa cccggcctcc cctcccctct ccctgcttgt gctgcacagg 420 ctgctctgtg agcacttcag ggtcctgtcc acggtgcaca cgcactcctc ggtcaagagc 480 gtgcctgaaa accttctcaa gtgctttgga gaacagaata aaaaacagcc ccgccaagac 540 tatcagctgg gattcacttt aatttggaag aatgtgccga agacgcagat gaaattcagc 600 atccagacga tgtgccccat cgaaggcgaa gggaacattg cacgtttctt gttctctctg 660 tttggccaga agcataatgc tgtcaacgca acccttatag atagctgggt agatattgcg 720 atttttcagt taaaagaggg aagcagtaaa gaaaaagccg ctgttttccg ctccatgaac 780 tctgctcttg ggaagagccc ttggctcgct gggaatgaac tcaccgtagc agacgtggtg 840 ctgtggtctg tactccagca gatcggaggc tgcagtgtga cagtgccagc caatgtgcag 900 aggtggatga ggtcttgtga aaacctggct cctttttcca gcgctaagga tccaccggtc 960 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 1020 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 1080 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg 1140 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac 1200 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 1260 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 1320 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 1380 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag 1440 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag 1500 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac 1560 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac 1620 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtaa 1680                                                                         1680 <210> 7 <211> 2367 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 7 atggtaaagc gtgagaaaaa tgtcatctat ggccctgagc ctctccatcc tttggaggat 60 ttgactgccg gcgaaatgct gtttcgtgct ctccgcaagc actctcattt gcctcaagcc 120 ttggtcgatg tggtcggcga tgaatctttg agctacaagg agttttttga ggcaaccgtc 180 ttgctggctc agtccctcca caattgtggc tacaagatga acgacgtcgt tagtatctgt 240 gctgaaaaca atacccgttt cttcattcca gtcatcgccg catggtatat cggtatgatc 300 gtggctccag tcaacgagag ctacattccc gacgaactgt gtaaagtcat gggtatctct 360 aagccacaga ttgtcttcac cactaagaat attctgaaca aagtcctgga agtccaaagc 420 cgcaccaact ttattaagcg tatcatcatc ttggacactg tggagaatat tcacggttgc 480 gaatctttgc ctaatttcat ctctcgctat tcagacggca acatcgcaaa ctttaaacca 540 ctccacttcg accctgtgga acaagttgca gccattctgt gtagcagcgg tactactgga 600 ctcccaaagg gagtcatgca gacccatcaa aacatttgcg tgcgtctgat ccatgctctc 660 gatccacgct acggcactca gctgattcct ggtgtcaccg tcttggtcta cttgcctttc 720 ttccatgctt tcggctttca tattactttg ggttacttta tggtcggtct ccgcgtgatt 780 atgttccgcc gttttgatca ggaggctttc ttgaaagcca tccaagatta tgaagtccgc 840 agtgtcatca acgtgcctag cgtgatcctg tttttgtcta agagcccact cgtggacaag 900 tacgacttgt cttcactgcg tgaattgtgt tgcggtgccg ctccactggc taaggaggtc 960 gctgaagtgg ccgccaaacg cttgaatctt ccagggattc gttgtggctt cggcctcacc 1020 gaatctacca gtgcgattat ccagactctc ggggatgagt ttaagagcgg ctctttgggc 1080 cgtgtcactc cactcatggc tgctaagatc gctgatcgcg aaactggtaa ggctttgggc 1140 ccgaaccaag tgggcgagct gtgtatcaaa ggccctatgg tgagcaaggg ttatgtcaat 1200 aacgttgaag ctaccaagga ggccatcgac gacgacggct ggttgcattc tggtgatttt 1260 ggatattacg acgaagatga gcatttttac gtcgtggatc gttacaagga gctgatcaaa 1320 tacaagggta gccaggttgc tccagctgag ttggaggaga ttctgttgaa aaatccatgc 1380 attcgcgatg tcgctgtggt cggcattcct gatctggagg ccggcgaact gccttctgct 1440 ttcgttgtca agcagcctgg tacagaaatt accgccaaag aagtgtatga ttacctggct 1500 gt; cgtaacgtaa caggcaaaat tacccgcaag gagctgttga aacaattgtt ggtgaaggcc 1620 ggcggtagcg ctaaggatcc accggtcgcc accatggtga gcaagggcga ggagctgttc 1680 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc 1740 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc 1800 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg 1860 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg 1920 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc 1980 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc 2040 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac 2100 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc 2160 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 2220 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc 2280 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 2340 atcactctcg gcatggacga gctgtaa 2367 <210> 8 <211> 1455 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 8 atggcggtgg aaggaggaat gaaatgtgtg aagttcttgc tctacgtcct cctgctggcc 60 ttttgcgcct gtgcagtggg actgattgcc gtgggtgtcg gggcacagct tgtcctgagt 120 cagaccataa tccagggggc tacccctggc tctctgttgc cagtggtcat catcgcagtg 180 ggtgtcttcc tcttcctggt ggcttttgtg ggctgctgcg gggcctgcaa ggagaactat 240 tgtcttatga tcacgtttgc catctttctg tctcttatca tgttggtgga ggtggccgca 300 gccattgctg gctatgtgtt tagagataag gtgatgtcag agtttaataa caacttccgg 360 cagcagatgg agaattaccc gaaaaacaac cacactgctt cgatcctgga caggatgcag 420 gcagatttta agtgctgtgg ggctgctaac tacacagatt gggagaaaat cccttccatg 480 tcgaagaacc gagtccccga ctcctgctgc attaatgtta ctgtgggctg tgggattaat 540 ttcaacgaga aggcgatcca taaggagggc tgtgtggaga agattggggg ctggctgagg 600 aaaaatgtgc tggtggtagc tgcagcagcc cttggaattg cttttgtcga ggttttggga 660 attgtctttg cctgctgcct cgtgaagagt atcagaagtg gctacgaggt gatgagcgct 720 aaggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg 780 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 840 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 900 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc 960 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 1020 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 1080 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 1140 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc 1200 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag 1260 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 1320 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac 1380 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc 1440 atggacgagc tgtaa 1455 <210> 9 <211> 1863 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 9 atgtggttct tgacaactct gctcctttgg gttccagttg atgggcaagt ggacaccaca 60 aaggcagtga tcactttgca gcctccatgg gtcagcgtgt tccaagagga aaccgtaacc 120 ttgcattgtg aggtgctcca tctgcctggg agcagctcta cacagtggtt tctcaatggc 180 acagccactc agacctcgac ccccagctac agaatcacct ctgccagtgt caatgacagt 240 ggtgaataca ggtgccagag aggtctctca gggcgaagtg accccataca gctggaaatc 300 cacagaggct ggctactact gcaggtctcc agcagagtct tcacggaagg agaacctctg 360 gccttgaggt gtcatgcgtg gaaggataag ctggtgaca atgtgcttta ctatcgaaat 420 ggcaaagcct ttaagttttt ccactggaat tctaacctca ccattctgaa aaccaacata 480 agtcacaatg gcacctacca ttgctcaggc atgggaaagc atcgctacac atcagcagga 540 atatctgtca ctgtgaaaga gctatttcca gctccagtgc tgaatgcatc tgtgacatcc 600 ccactcctgg aggggaatct ggtcaccctg agctgtgaaa caaagttgct cttgcagagg 660 cctggtttgc agctttactt ctccttctac atgggagag agaccctgcg aggcaggaac 720 acatcctctg aataccaaat actaactgct agaagagaag actctgggtt atactggtgc 780 gaggctgcca cagaggatgg aaatgtcctt aagcgcagcc ctgagttgga gcttcaagtg 840 cttggcctcc agttaccaac tcctgtctgg tttcatgtcc ttttctatct ggcagtggga 900 ataatgtttt tagtgaacac tgttctctgg gtgacaatac gtaaagaact gaaaagaaag 960 aaaaagtggg atttagaaat ctctttggat tctggtcatg agaagaaggt aatttccagc 1020 cttcaagaag acagacattt agaagaagag ctgaaatgtc aggaacaaaa agaagaacag 1080 ctgcaggaag gggtgcaccg gaaggagccc cagggggcca cgagcgctaa ggatccaccg 1140 gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 1200 ggctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 1260 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 1320 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac 1380 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 1440 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 1500 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 1560 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 1620 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 1680 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 1740 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 1800 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 1860 taa 1863 <210> 10 <211> 1650 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 10 atggctacct ctcgatatga gccagtggct gaaattggtg tcggtgccta tgggacagtg 60 tacaaggccc gtgatcccca cagtggccac tttgtggccc tcaagagtgt gagagtcccc 120 aatggaggag gaggtggagg aggccttccc atcagcacag ttcgtgaggt ggctttactg 180 aggcgactgg aggcttttga gcatcccaat gttgtccggc tgatggacgt ctgtgccaca 240 tcccgaactg accgggagat caaggtaacc ctggtgtttg agcatgtaga ccaggaccta 300 aggacatatc tggacaaggc acccccacca ggcttgccag ccgaaacgat caaggatctg 360 atgcgccagt ttctaagagg cctagatttc cttcatgcca attgcatcgt tcaccgagat 420 ctgaagccag agaacattct ggtgacaagt ggtggaacag tcaagctggc tgactttggc 480 ctggccagaa tctacagcta ccagatggca cttacacccg tggttgttac actctggtac 540 cgagctcccg aagttcttct gcagtccaca tatgcaacac ctgtggacat gtggagtgtt 600 ggctgtatct ttgcagagat gtttcgtcga aagcctctct tctgtggaaa ctctgaagcc 660 gaccagttgg gcaaaatctt tgacctgatt gggctgcctc cagaggatga ctggcctcga 720 gatgtatccc tgccccgtgg agcctttccc cccagagggc cccgcccagt gcagtcggtg 780 gtacctgaga tggaggagtc gggagcacag ctgctgctgg aaatgctgac ttttaaccca 840 cacaagcgaa tctctgcctt tcgagctctg cagcactctt atctacataa ggatgaaggt 900 aatccggaga gcgctaagga tccaccggtc gccaccatgg tgagcaaggg cgaggagctg 960 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 1020 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 1080 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 1140 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 1200 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 1260 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 1320 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 1380 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 1440 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 1500 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 1560 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 1620 gggatcactc tcggcatgga cgagctgtaa 1650 <210> 11 <211> 2103 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 11 atggatttcc tttgggcgtt ggaaaccccg cagacagcca cgacgatgcc cctcaacgtg 60 aacttcacca acaggaacta tgacctcgac tacgactccg tacagcccta tttcatctgc 120 gacgaggaag agaatttcta tcaccagcaa cagcagagcg agctgcagcc gcccgcgccc 180 agtgaggata tctggaagaa attcgagctg cttcccaccc cgcccctgtc cccgagccgc 240 cgctccgggc tctgctctcc atcctatgtt gcggtcgcta cgtccttctc cccaagggaa 300 gacgatgacg gcggcggtgg caacttctcc accgccgatc agctggagat gatgaccgag 360 ttacttggag gagacatggt gaaccagagc ttcatctgcg atcctgacga cgagaccttc 420 atcaagaaca tcatcatcca ggactgtatg tggagcggtt tctcagccgc tgccaagctg 480 gtctcggaga agctggcctc ctaccaggct gcgcgcaaag acagcaccag cctgagcccc 540 gcccgcgggc acagcgtctg ctccacctcc agcctgtacc tgcaggacct caccgccgcc 600 gcgtccgagt gcattgaccc ctcagtggtc tttccctacc cgctcaacga cagcagctcg 660 cccaaatcct gtacctcgtc cgattccacg gccttctctc cttcctcgga ctcgctgctg 720 tcctccgagt cctccccacg ggccagccct gagcccctag tgctgcatga ggagacaccg 780 cccaccacca gcagcgactc tgaagaagag caagaagatg aggaagaaat tgatgtggtg 840 tctgtggaga agaggcaaac ccctgccaag aggtcggagt cgggctcatc tccatcccga 900 ggccacagca aacctccgca cagcccactg gtcctcaaga ggtgccacgt ctccactcac 960 cagcacaact acgccgcacc cccctccaca aggaaggact atccagctgc caagagggcc 1020 aagttggaca gtggcagggt cctgaagcag atcagcaaca accgcaagtg ctccagcccc 1080 aggtcctcag acacggagga aaacgacaag aggcggacac acaacgtctt ggaacgtcag 1140 aggaggaacg agctgaagcg cagctttttt gccctgcgtg accagatccc tgaattggaa 1200 aacaacgaaa aggcccccaa ggtagtgatc ctcaaaaaag ccaccgccta catcctgtcc 1260 attcaagcag acgagcacaa gctcacctct gaaaaggact tattgaggaa acgacgagaa 1320 cagttgaaac acaaactcga acagcttcga aactctggtg caagcgctaa ggatccaccg 1380 gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 1440 ggctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 1500 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 1560 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac 1620 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 1680 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 1740 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 1800 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 1860 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 1920 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 1980 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 2040 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 2100 taa 2103 <210> 12 <211> 1473 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 12 atgccgatgt accaggtaaa gccctatcac gggggcggcg cgcctctccg tgtggagctt 60 cccacctgca tgtaccggct ccccaacgtg cacggcagga gctacggccc agcgccgggc 120 gctggccacg tgcaggatta cggggcgctg aaagacatcg tgatcaacgc aaacccggcc 180 tcccctcccc tctccctgct tgtgctgcac aggctgctct gtgagcactt cagggtcctg 240 tccacggtgc acacgcactc ctcggtcaag agcgtgcctg aaaaccttct caagtgcttt 300 ggagaacaga ataaaaaaca gccccgccaa gactatcagc tgggattcac tttaatttgg 360 aagaatgtgc cgaagacgca gatgaaattc agcatccaga cgatgtgccc catcgaaggc 420 gaagggaaca ttgcacgttt cttgttctct ctgtttggcc agaagcataa tgctgtcaac 480 gcaaccctta tagatagctg ggtagatatt gcgatttttc agttaaaaga gggaagcagt 540 ccgctgttc gctgggaatg aactcaccgt agcagacgtg gtgctgtggt ctgtactcca gcagatcgga 660 ggctgcagtg tgacagtgcc agccaatgtg cagaggtgga tgaggtcttg tgaaaacctg 720 gctccttttt ccagcgctaa ggatccaccg gtcgccacca tggtgagcaa gggcgaggag 780 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 840 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc 900 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac 960 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 1020 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac 1080 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 1140 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac 1200 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag 1260 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 1320 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc 1380 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 1440 gccgggatca ctctcggcat ggacgagctg taa 1473 <210> 13 <211> 1854 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 13 atggctgcaa cctgtgagat tagcaacatt tttagcaact acttcagtgc gatgtacagc 60 tcggaggact ccaccctggc ctctgttccc cctgctgcca cctttggggc cgatgacttg 120 gtactgaccc tgagcaaccc ccagatgtca ttggagggta cagagaaggc cagctggttg 180 ggggaacagc cccagttctg gtcgaagacg caggttctgg actggatcag ctaccaagtg 240 gagaagaaca agtacgacgc aagcgccatt gacttctcac gatgtgacat ggatggcgcc 300 accctctgca attgtgccct tgaggagctg cgtctggtct ttgggcctct gggggaccaa 360 ctccatgccc agctgcgaga cctcacttcc agctcttctg atgagctcag ttggatcatt 420 gagctgctgg agaaggatgg catggccttc caggaggccc tagacccagg gccctttgac 480 cagggcagcc cctttgccca ggagctgctg gacgacggtc agcaagccag cccctaccac 540 cccggcagct gtggcgcagg agccccctcc cctggcagct ctgacgtctc caccgcaggg 600 actggtgctt ctcggagctc ccactcctca gactccggtg gaagtgacgt ggacctggat 660 cccactgatg gcaagctctt ccccagcgat ggttttcgtg actgcaagaa gggggatccc 720 aagcacggga agcggaaacg aggccggccc cgaaagctga gcaaagagta ctgggactgt 780 ctcgagggca agaagagcaa gcacgcgccc agaggcaccc acctgtggga gttcatccgg 840 gacatcctca tccacccgga gctcaacgag ggcctcatga agtgggagaa tcggcatgaa 900 ggcgtcttca agttcctgcg ctccgaggct gtggcccaac tatggggcca aaagaaaaag 960 aagacca tgacctacga gaagctgagc cgggccatga ggtactacta caaacgggag 1020 atcctggaac gggtggatgg ccggcgactc gtctacaagt ttggcaaaaa ctcaagcggc 1080 tggaaggagg aagaggttct ccagagtcgg aacagcgcta aggatccacc ggtcgccacc 1140 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 1200 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 1260 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 1320 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 1380 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 1440 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 1500 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 1560 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 1620 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 1680 gccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 1740 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 1800 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtaa 1854 <210> 14 <211> 1230 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 14 atggcggacg aggagaagct gccgcccggc tgggagaagc gcatgagccg cagctcaggc 60 cgagtgtact acttcaacca catcactaac gccagccagt gggagcggcc cagcggcaac 120 agcagcagtg gtggcaaaaa cgggcagggg gagcctgcca gggtccgctg ctcgcacctg 180 ctggtgaagc acagccagtc acggcggccc tcgtcctggc ggcaggagaa gatcacccgg 240 accaaggagg aggccctgga gctgatcaac ggctacatcc agaagatcaa gtcgggagag 300 gaggactttg agtctctggc ctcacagttc agcgactgca gctcagccaa ggccagggga 360 gacctgggtg ccttcagcag aggtcagatg cagaagccat ttgaagacgc ctcgtttgcg 420 ctgcggacgg gggagatgag cgggcccgtg ttcacggatt ccggcatcca catcatcctc 480 cgcactgaga gcgctaagga tccaccggtc gccaccatgg tgagcaaggg cgaggagctg 540 ctggcggcg agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 660 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 720 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 780 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 840 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 900 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 960 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 1020 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 1080 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 1140 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 1200 gggatcactc tcggcatgga cgagctgtaa 1230 <210> 15 <211> 1335 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 15 cgccaggcag 60 gcggagcacc ccaagccctc ggcctgcagg aacctcttcg gcccggtgga ccacgaagag 120 ttaacccggg acttggagaa gcactgcaga gacatggaag aggcgagcca gcgcaagtgg 180 aatttcgatt ttcagaatca caaaccccta gagggcaagt acgagtggca agaggtggag 240 aagggcagct tgcccgagtt ctactacaga cccccgcggc cccccaaagg tgcctgcaag 300 gtgccggcgc aggagagcca ggatgtcagc gggagccgcc cggcggcgcc tttaattggg 360 gctccggcta actctgagga cacgcatttg gtggacccaa agactgatcc gtcggacagc 420 cagacggggt tagcggagca atgcgcagga ataaggaagc gacctgcaac cgacgattct 480 tctactcaaa acaaaagagc caacagaaca gaagaaaatg tttcagacgg ttccccaaat 540 gccggttctg tggagcagac gcccaagaag cctggcctca gaagacgtca aacgagcgct 600 aaggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg 660 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 720 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 780 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc 840 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 900 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 960 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 1020 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc 1080 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag 1140 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 1200 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac 1260 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc 1320 atggacgagc tgtaa 1335 <210> 16 <211> 1479 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 16 atggagtctg gcaagatggc gtctcccaag agcatgccga aagatgcaca gatgatggca 60 caaatcctga aggatatggg gattacagaa tatgagccaa gagttataaa tcagatgctg 120 gagtttgcct tccgatatgt gacaacaatt ctagatgatg ctaaaattta ttccagccat 180 gctaagaaag ctactgttga tgcagatgac gtacgactgg caatccagtg ccgtgctgac 240 cagtctttca cttctccacc cccgagagat ttcttattag atattgcacg acaaagaaat 300 caaacccctt tgccactgat caagccatat tcaggtccta gattgccacc tgataggtat 360 tgcttaactg ccccaaacta taggcttaag tctttacaga aaaaggcacc tgctcctgca 420 ggaagaataa cagttccaag gttaagtgtt ggttcagttt ctagtagacc aagtactccc 480 accctaggta caccgactcc acaaaccatg tctgtttcaa ctaaagtagg caccccaatg 540 tccctcacag gacagaggtt tacagtacag atgcctgctt ctcagtcccc tgctgtaaaa 600 gcttctatac ctgcaacatc aacagttcag aatgttctta ttaatccatc gttaattggg 660 tccaaaaaca ttcttattac tactaatatg gtatcacaga atacagccga gtcagcaaat 720 gcactgaaac ggaaacgtag cgctaaggat ccaccggtcg ccaccatggt gagcaagggc 780 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 840 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 900 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg 960 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc 1020 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc 1080 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag 1140 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac 1200 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac 1260 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 1320 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag 1380 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 1440 accgccgccg ggatcactct cggcatggac gagctgtaa 1479

Claims (11)

The R-box protein 1 and the translational fusion constructs in which the C-terminal of SEQ ID NO: 1 is removed are treated with a substrate protein, A method for degrading a substrate protein. delete 2. The method according to claim 1, wherein the substrate protein is a protein existing in a cell nucleus or cytosol. The method of claim 1, wherein the substrate protein is a fluorescent protein. 2. The method of claim 1, wherein the nanobody binds to a protein present in a cell nucleus or cytosol. 2. The method of claim 1, wherein the complex of the nanobodies and the C-terminal removed ring-box protein 1 is overexpressed. A composition for degrading a substrate protein comprising a ring-box protein 1 (RING-box protein 1) having a C-terminal removed in SEQ ID NO: 1 and translational fusion constructs of a nano-body. delete 8. The composition of claim 7, wherein the substrate protein is a protein present in a cell nucleus or cytosol. 8. The composition of claim 7, wherein the substrate protein is a fluorescent protein. 8. The composition of claim 7, wherein the nanobody binds to a protein present in a cell nucleus or cytosol.

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