CN114133439A

CN114133439A - Mutant xSUMO, mutant xE1, and xSUMO-xE1 combination mutants, and related products and uses thereof

Info

Publication number: CN114133439A
Application number: CN202111222826.9A
Authority: CN
Inventors: 赵博; 叶琳; 刘金钊; 康晓彤; 李贞�; 张湘奇; 王亚楠
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-10-20
Filing date: 2021-10-20
Publication date: 2022-03-04
Anticipated expiration: 2041-10-20
Also published as: CN114133439B

Abstract

The invention discloses a mutant xSUMO, a mutant xE1 and an xSUMO-xE1 combined mutant and related products and application thereof, wherein the mutant xSUMO is a protein as shown in the following (1) or (2): (1) a protein consisting of an amino acid sequence as shown in SEQ ID No.1 or SEQ ID No. 3; (2) and (2) the protein which is derived from the protein (1) and has the same function by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO. 3. The invention constructs mutant xSUMO, mutant xE1 and xSUMO-xE1 combined mutant, can specifically recognize the substrate of SUMO conjugated enzyme E2(Ubc9) or E3 substrate, and can be used as target protein for screening anti-neurodegenerative diseases or anti-cancer drugs.

Description

Mutant xSUMO, mutant xE1, and xSUMO-xE1 combination mutants, and related products and uses thereof

Technical Field

The invention relates to the technical field of biology, in particular to a mutant xSUMO, a mutant xE1 and a xSUMO-xE1 combined mutant and related products and application thereof.

Background

Ubiquitin-like protein modifying molecule (SUMO) is a very important Small molecular weight protein (12 kDa) present in eukaryotic cells. SUMO, as a post-translational modification molecule, can be delivered to more than 1000 substrate proteins.

Like Ubiquitin (UB), SUMO needs to be delivered to a substrate via a series of specific enzymes, a process called SUMO methylation. SUMO expressed in cells is initially present as a precursor, which upon specific proteolytic cleavage exposes the C-terminal bisglycine residue, which becomes functional SUMO. In the presence of ATP, SUMO is catalytically activated by SUMO activating enzyme E1(SAE), E1 is a dimer formed by the isomerization of Sae1 and Uba2, and the exposed C-terminal bisglycine residue forms a thioester bond with the cysteine residue of Uba 2. Subsequently, the activated SUMO was transferred from E1 to the cysteine residue of the only SUMO binding enzyme E2(SUMO conjugation enzyme, Ubc9) in the cell to form a second thioester bond. SUMO ligase E3(SUMO ligase enzyme) then binds to and catalyzes the attachment of SUMO to the substrate protein.

Unlike Ub, SUMO has two pathways for delivery to the substrate protein: the first is that in the absence of SUMO ligase E3, Ubc9 binds directly to the substrate protein, delivers SUMO to the substrate and catalyzes the C-terminal bisglycine residue of SUMO to form an isopeptide bond with the lysine epsilon-amino group of the substrate protein, such substrate being referred to as the specific substrate of Ubc 9; the second pathway is that in the presence of SUMO ligase E3, Ubc9 first binds to SUMO ligase E3 and delivers SUMO directly to the substrate protein, which is referred to as the specific substrate for E3. To date, there is no clear literature reporting which of these substrates are specific substrates for Ubc9 and which are specific substrates for E3.

CN110845596B discloses that Western Blot detection of mutant xSUMO5(R70E, Y91A, E93R), xSUMO5 and wild type wtSAE (namely wild type activating enzyme E1) shows that xSUMO5 and wtSAE have no reactivity and have no direct effect. Subsequent studies have found that xSUMO5 can still be delivered to wild-type binding enzyme E2 by wild-type activating enzyme E1 in the presence of wild-type binding enzyme E2.

Disclosure of Invention

The invention aims to provide a mutant xSUMO, a mutant xE1 and an xSUMO-xE1 combined mutant and related products and application thereof, which are used for constructing an orthogonal SUMO transfer pathway and solve the problems that in the prior art, xSUMO5 has weak binding force with wild-type activating enzyme E1 even though the wild-type activating enzyme E1 cannot detect the direct action, so that xSUMO5 can still be transferred to the wild-type binding enzyme E2 through the wild-type activating enzyme E1, and the specific substrates of E3 and Ubc9 cannot be effectively distinguished.

One of the objects of the present invention is to provide a mutant xSUMO, which is a protein of the following (1) or (2):

(1) a protein consisting of an amino acid sequence as shown in SEQ ID No.1 or SEQ ID No. 3;

(2) and (2) the protein which is derived from the protein (1) and has the same function by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO. 3.

The invention also aims to provide a biological material related to the mutant xSUMO, which comprises any one of the following substances:

a) a polynucleotide encoding a mutant xSUMO as described;

b) a recombinant expression vector comprising the polynucleotide of a);

c) a biological engineering bacterium containing the polynucleotide of a) or a biological engineering bacterium containing the recombinant expression vector of b).

According to the technical scheme of the application, in a), the polynucleotide comprises a nucleotide sequence shown as SEQ ID NO.2 or SEQ ID NO. 4.

The recombinant expression vector of the present invention is constructed by inserting the polynucleotide into the multiple cloning site of an expression vector, which is generally referred to as a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as adenovirus, retrovirus or other vectors, as is well known in the art.

The bioengineering bacteria of the invention comprise the recombinant expression vector or the polynucleotide integrated in the genome. The bioengineered bacteria can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells.

The invention also aims to provide a mutant xE1, wherein the mutant is a SUMO activating enzyme E1 subunit Uba2 mutant, and the mutant is a protein as shown in the following (1) or (2):

(1) a protein consisting of an amino acid sequence shown as SEQ ID No. 5;

(2) and (2) the protein which is derived from the protein (1) and has the same function, and the amino acid sequence shown as SEQ ID NO.5 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues.

SUMO activating enzyme E1(SUMO activating enzyme, SAE) is a dimer formed by the isomerization of subunits Sae1 and Uba 2. Wherein, the amino acid sequence and the nucleotide sequence of the subunit Sae1 are respectively shown as SEQ ID NO.8 and SEQ ID NO. 9.

It is a fourth object of the present invention to provide biological material related to the mutant xE1 as described, including any of the following:

a) a polynucleotide encoding a mutant xE1 as described;

b) a recombinant expression vector comprising the polynucleotide of a);

According to one embodiment of the present invention, the polynucleotide of a) comprises a nucleotide sequence shown as SEQ ID NO. 6.

The fifth purpose of the invention is to provide an xSUMO-xE1 combination mutant, wherein the xSUMO-xE1 combination mutant comprises the mutant xSUMO;

and/or, the xSUMO-xE1 combination mutant comprises the mutant xE 1.

It is a further object of the present invention to provide biomaterials related to the xSUMO-xE1 combination mutants as described, including any of the following:

a) polynucleotides encoding the xSUMO-xE1 combination mutants as described;

b) a recombinant expression vector comprising the polynucleotide of a);

The seventh object of the present invention is the use of providing a mutant xSUMO as described or a biomaterial related to mutant xSUMO as described or mutant xE1 as described or a biomaterial related to mutant xE1 as described or a combination mutant of xSUMO-xE1 as described or a biomaterial related to a combination mutant of xSUMO-xE1 as described for identifying a substrate for Ubc9 or a substrate for E3.

The eighth object of the present invention is to provide the use of the mutant xSUMO or the biological material related to the mutant xSUMO as described above or the mutant xE1 as described above or the biological material related to mutant xE1 as described above or the xSUMO-xE1 combination mutant as described above or the biological material related to xSUMO-xE1 combination mutant as described above as a target protein in screening anti-neurodegenerative diseases or anti-cancer drugs.

The mutant xSUMO or the mutant xE1 or the xSUMO-xE1 combined mutant can screen substrate proteins which are specifically combined with Ubc9 or SUMO ligase E3, the substrate proteins are related to neurodegenerative diseases such as Alzheimer's disease or Parkinson and cancer, and E3 corresponding to the substrate proteins is studied in depth, so that E3 can be a potential drug target.

In this application, SUMO activating enzyme E1(SAE) is designated E1, SUMO-binding enzyme E2(Ubc9) is designated E2, and SUMO ligase E3 is designated E3.

The invention has the following beneficial effects:

1) according to the invention, firstly, all mutants including mutant xSUMO, mutant xE (xE1, xE2, xE3) and xSUMO-xE combined mutant (xSUMO-xE1-xE2 or xSUMO-xE1-xE2-xE3) are constructed at a molecular level by a phage display technology, and the vitality between the mutants is detected by protein level SUMO, and the vitality between the mutants and specific substrates of wild type wtSUMO, wild type wtE1, wild type E2 and E2 is detected, so that a method for specifically identifying a SUMO-binding enzyme E2(Ubc9) substrate is provided, and a theoretical basis is laid for identifying a SUMO ligase E3(E3) substrate.

2) The technical scheme in the application can distinguish the specific substrate of the SUMO combined enzyme E2(Ubc9) for the first time, is favorable for deepening the understanding of the whole SUMO signal transduction path, and lays a theoretical foundation for further discovering a new substrate of a specific SUMO ligase E3(E3) and developing a medicine aiming at E3.

Drawings

FIG. 1 is a graph showing sequencing comparison results of the mutant xE1-1 constructed in example 1 of the present invention.

FIG. 2 is a graph showing sequencing comparison results of the mutant xE1-1 constructed in example 1 of the present invention.

FIG. 3 is a Western blot analysis chart of the mutant xE1-1 of example 1 of the present invention.

FIG. 4 is a Western blot analysis chart of the mutant xE1-2 of example 1 of the present invention.

FIG. 5 is a Western blot analysis chart of the mutant xE1-3 of example 1 of the present invention.

FIG. 6 shows the electrophoretograms of the cleaved products of mutant xSUMO-1, mutant xSUMO-2 and plasmid PET28a in example 2 of the present invention.

FIG. 7 is a chart showing the alignment of mutant xSUMO-1 and mutant xSUMO5 in example 2 of the present invention.

FIG. 8 is a chart showing the alignment of mutant xSUMO-2 and mutant xSUMO5 in example 2 of the present invention.

FIG. 9 shows electrophoretograms of supernatant, flow-through and eluate obtained by purifying mutant xSUMO-1 and mutant xSUMO-2 in example 2 of the present invention by Ni-Beads affinity chromatography.

FIG. 10 is a graph showing the Western blot detection results of the combination mutant of xSUMO5-xE1-1 in example 2 of the present invention.

FIG. 11 is a graph showing the Western blot detection results of the combination mutant of xSUMO5-xE1-1 in example 2 of the present invention after adding wild-type E2.

FIG. 12 is a graph showing the results of Western blot detection of mutant xSUMO-1 and mutant xSUMO-2 in example 2 of the present invention.

FIG. 13 is a graph showing the results of Western blot detection of RanGTP enzyme agonist protein (RanGAP) obtained by adding a specific substrate of E2 to mutant xSUMO-1 and mutant xSUMO-2 in example 2 of the present invention.

FIG. 14 shows a schematic of the orthogonal ubiquitination delivery pathway.

FIG. 15 is a graph showing a standard curve when the protein concentration is measured by the BCA method in example 2 of the present invention.

The drawings in fig. 10 to 13 are explained as follows

xE1 represents the mutant xE1-1 obtained in example 1.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Applicants previously constructed a completely new orthogonal ubiquitination pathway in cells to identify the substrate of E3, and the ubiquitination pathway is shown in fig. 14. A mutant xUB, a mutant xE1, a mutant xE2 and a mutant xE3 in the ubiquitination pathway are constructed, the mutants do not react with any wild type E1, E2 and E3 or wild type UB in a cell, only the mutants react, so that an ubiquitin pathway which is not interfered with the wild type pathway is generated in the cell, the mutant xUB is transferred to a substrate by the pathway, and the protein to which xUB is combined can be determined by detecting tag carried on xUB, and then the protein is the substrate of the specific E3, and then the substrates are verified by adopting a cytobiological means.

According to the invention, based on the previous research foundation, an orthogonal SUMO (mito for short) delivery pathway is constructed in cells, namely, firstly, mutants xSUMO and xE1 are constructed, the specific substrate of Ubc9 is determined by constructing an xSUMO-xE1 combined mutant and an xE1-xE2 combined mutant, and the specific substrate of E3 is determined by additionally constructing an xE2-xE3 combined mutant.

In the present invention, the mutant xE1 was constructed in the OST, which reacts only with mutant xSUMO5, but not with wild-type wtSUMO. Depending on the mutation site of xSUMO5, the introduction of the corresponding mutation site in E1 restored the viability of xSUMO 5. By analyzing the structures of wtSUMO and wtE1, the key sites in E1 that acted on the xSUMO5 mutation site were determined and mutations were made at these sites to obtain mutant xE1 that reacted with mutant xSUMO5, and mutant xE1 that did not react with wild-type wtSUMO.

In the invention, mutant xSUMO-1 and mutant xSUMO-2 which do not react with wild-type wtE1 in OST are constructed in the second aspect, and mutant xSUMO-1 and mutant xSUMO-2 both react with mutant xE1 constructed in the first aspect and are not transmitted to wild-type E2 through wild-type wtE1, but can be transmitted to wild-type E2 through mutant xE1, and wild-type wtSUMO and wild-type wtE1 do not interfere with OST pathway.

In the invention, an orthogonal xSUMO-xE1 combined mutant is constructed in the third aspect, and the specific substrate of Ubc9 can be identified by transferring the mutant xSUMO to a wild type E2 through a mutant xE1 and then transferring the mutant xSUMO to an E2 specific substrate RanGAP from a wild type E2; the specific substrate of E3 can be further determined by adding construction such as an xE2-xE3 combined mutant.

In the examples of the present application, the materials are as follows:

2 xTaq PCR MasterMix kit, purchased from Shaoxins Biotechnology Ltd;

plasmid Mimi Kit, Gel Extraction Kit, purchased from Omega bio-tech;

BCA protein content detection kit purchased from Keyji organisms;

LB liquid medium: 10g of Tryptone (Tryptone), 5g of Yeast extract (Yeast extract), and 10g of NaCl were weighed into a 1L glass bottle, and ddH was added₂And O is metered to 1L.

LB solid medium: 5g Tryptone (Tryptone), 2.5g Yeast extract (Yeast extract), 5g NaCl, 5g Agar (Agar) were weighed into1L round bottom flask, add ddH₂And O is metered to 500 mL.

SOC liquid medium: 10g of Tryptone (Tryptone), 2.5g of Yeast extract (Yeast extract) and 0.25g of NaCl were weighed into a 500mL glass vial and added with ddH₂Dissolving O, adding 5mL of 0.25M KCl solution, 2M MgCl₂2.5mL of the solution is added to 490mL of the solution, and the solution is sterilized at high temperature and high pressure. After cooling at room temperature, 10mL of 1M Glucose (Glucose) solution after filtration sterilization through a 0.22 μ M filter membrane was added, mixed well, and stored at 4 ℃.

50X TAE buffer: 242.2g Tris base and 37.2g Na were weighed out₂EDTA·2H₂O into a 1L glass bottle, 800mL ddH was added₂O, placing on a stirrer, fully dissolving the solid under the stirring of a magnet rotor, adding 57.1mL of glacial acetic acid, fully mixing, and adding ddH₂And (4) metering the volume of O to 1L, and storing at room temperature. When in use, ddH is added₂O diluted 50-fold to 1 × TAE buffer.

DNA agarose gel (1%): 1g of agarose powder was weighed into a small shake flask, 1mL of 1 × TAE buffer was added, and the mixture was thoroughly dissolved by heating with a high fire in a microwave oven for 2 min. Cooling to 50 deg.C, adding 10 μ L of Dured dye (10000X), mixing, pouring into DNA gel tank, immediately inserting comb with appropriate pore size and pore number, and standing for about 0.5 hr until solidification.

EXAMPLE 1 construction of mutant xE1

In this example, mutant xE1 was constructed and expression and purification of mutant xE1 were performed using E1 as a study. Comprises the following steps:

1. construction of mutant xE1

Designing an upstream primer and a downstream primer containing corresponding mutation points, obtaining a front fragment and a rear fragment of xE1 through PCR by using the corresponding primers, and then integrating the front fragment and the rear fragment together through overlap PCR to obtain a gene fragment of xE 1; the construct was incorporated into pETDuet plasmid to give recombinant plasmid pETDuet-xE1, which was then electrotransformed into XL-1-Blue competent cells.

(1) PCR amplification with the extracted cDNA as template

The nucleotide sequence of xSUMO5 includes a sequence shown in SEQ ID NO.7, 3 different mutants xE1-1, xE1-2 and xE1-3 are sequentially designed based on the information of the mutation sites, and the mutation sites and primers are shown in tables 1 and 2.

TABLE 1

Mutant	PCR template	Primer	Restriction enzyme
				xE1-1	pETDuet-wtE1	LIN9(F),LIN11(OR),LIN12(OF),LIN10(R)	NheI,MfeI
xE1-2	pETDuet-xE1-1	LIN13(F),LIN14(OR),LIN15(OF),LIN16(R)	StuI,XhoI
				xE1-3	pETDuet-xE1-2	LIN13(F),LIN17(OR),LIN18(OF),LIN16(R)	StuI,XhoI

Note: f refers to an upstream primer of an xE1 fragment, and R refers to a downstream primer of an xE1 fragment; OR refers to a downstream primer OF an XE1 front-section overlap fragment, and OF refers to an upstream primer OF an XE1 rear-section overlap fragment. The plasmid template for xE1-1 was pETDuet-wtE1, forming pETDuet-xE 1-1. The plasmid template for xE1-2 was pETDuet-xE1, forming pETDuet-xE 1-2. The plasmid template for xE1-3 was pETDuet-xE1-2, forming pETDuet-xE 1-3.

TABLE 2

The reaction system is shown in Table 3. Adding and uniformly mixing samples required by a reaction system, and then putting the mixture into a PCR instrument for amplification, wherein the procedure is as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 58 deg.C for 30s, extension at 72 deg.C for 1min, reacting for 35 cycles, extension at 72 deg.C for 5min, and temporary storage at 4 deg.C.

TABLE 3

(2) Gel electrophoresis

The PCR amplification product was electrophoresed (1% agarose gel prepared according to the molecular weight of DNA) at 115V for 30 min. Observing in an ultraviolet imager after the glue running is finished, cutting a target band (about 300bp), and then recovering and purifying a PCR product by using an Omega Gel extraction Kit, wherein the specific operation is carried out according to the instruction; finally, the concentration of the PCR product was determined by Nanodrop.

(3) The PCR products of the mutants xE1-1, xE1-2 and xE1-3 obtained in the step (1) and the plasmid pETDuet are respectively subjected to enzyme digestion by restriction enzymes StuI and XhoI

The restriction enzyme was digested with the restriction enzyme kit from NEB, and the specific procedures were performed according to the instructions. The cleavage reaction system is shown in Table 4. The reaction system is reacted in water bath at 37 ℃ for 3 h.

TABLE 4

Carrying out 1% agarose Gel electrophoresis on the enzyme digestion product, cutting a target band under an ultraviolet imager after the electrophoresis is finished, and then recovering and purifying a PCR product by using an Omega Gel extraction Kit, wherein the specific operation is carried out according to the instruction; and finally, measuring the concentration of the PCR product by using a micro spectrophotometer.

(4) Enzyme linked reaction

The digested fragments and digested plasmids were ligated using T4 DNA ligase (Thermo Scientific) in a 10. mu.L ligation system as follows: 10. mu.L of XE1-1, XE1-2 or XE1-3 fragment, 1. mu.L of pETDuet fragment, 1. mu.L of 10x T4 buffer, 1. mu.L of T4 Ligase. After mixing evenly, reacting for 1h at room temperature to obtain an enzyme-linked reaction product.

(5) Construction of recombinant plasmid

Taking 3 mu L of the enzyme-linked reaction product obtained in the step (4), and electrically transforming the enzyme-linked reaction product into XL-1-Blue competent cells. The method comprises the following specific steps:

mu.L of the ligation product was carefully mixed with about 50. mu.L of XL-1-Blue competent cells, iced for 30min, heat-shocked at 42 ℃ for 90s, then placed at 4 ℃ for resuscitation for 2min, 500. mu.L of SOC medium was added, shake-cultured at 37 ℃ for 1h, 400. mu.L of resuscitation fluid was spread on LB solid medium containing 100. mu. mol/L ampicillin, and incubated at 37 ℃ overnight.

On day 2, a single colony was picked up in 5mL of LB liquid medium containing 100. mu. mol/L ampicillin, and shaken at 37 ℃ and 220 r/min.

On the 3 rd day, 800 μ L of bacterial liquid and 200 μ L of 50% glycerol are mixed to prepare strains, and the residual bacterial liquid is subjected to miniprep of plasmid DNA by using a plasmid miniprep kit; the concentration of the recombinant plasmid is determined by using Nanodrop; and (3) sequencing the recombinant plasmid (Jinzhi), and then comparing a sequencing result with a template sequence to determine whether the target sequence is constructed successfully.

FIGS. 1 and 2 are graphs comparing the sequencing results of the constructed mutant xE 1-1.

As can be seen from FIGS. 1 and 2, the four mutation points (E157R, D435R, R119E, Y159E) of mutant xE1-1 were all correctly introduced, and no other mutation points were introduced, indicating that recombinant plasmid pETDuet-xE1-1 was successfully constructed.

2. Protein expression and purification of mutant xE1

2.1 prokaryotic expression

Using mutant xE1-1 as an example, 3. mu.L of the recombinant plasmid pETDuet-xE1-1 successfully constructed in step 1 was carefully mixed with 50. mu.L of BL21(DE3) competent cells, subjected to chemical transformation, and finally 400. mu.L of the resuscitative fluid was spread on an ampicillin plate and incubated overnight in a bacterial incubator at 37 ℃. After overnight, one single clone was picked up and put into 5mL of LB liquid medium containing ampicillin, shaken overnight at 37 ℃, and then transferred into 1L of LB liquid medium containing ampicillin, shaken at 37 ℃ until the bacterial density OD600 became 0.6-0.8, added with 1mL of IPTG (1M), and induced overnight at 16 ℃ for 16 hours.

Prokaryotic expression of mutant xE1-2 and mutant xE1-3 is the same as mutant xE 1-1.

2.2 protein purification

Taking mutant xE1-1 as an example, taking 1L of overnight induced bacterial liquid obtained in step 2.1, and centrifuging at 8000r/min for 10 min; the precipitate was resuspended in Lysis buffer, the cells were lysed thoroughly using a high pressure disruptor, the resulting lysate was centrifuged at 12000r/min for 30min, and the supernatant was collected.

40mL of the supernatant was mixed with 1mL of Ni-beads (Biorad) at 4 ℃ and the mixture was applied to a column and rinsed with Lysis Buffer, leaving 40mL of the flow-through. Then washed three times with Wash Buffer, and finally eluted with 3mL of Elution Buffer, and 15mL of eluate was retained. Dialyzed overnight at 4 ℃ using a 3.5KD dialysis bag.

The proteins of mutant xE1-2 and mutant xE1-3 were purified from mutant xE 1-1.

2.3 Activity assay

The activity of the mutants xE1-1, xE1-2 and xE1-3 was verified by using an in vitro enzyme-linked reaction in combination with Western blot. The in vitro enzyme-linked reaction system is shown in Table 5, and the reaction is carried out at 37 ℃ for 1 h.

TABLE 5

Species of	Concentration of
		wtSUMO or xSUMO5	4μM
wtE1 or xE1-1 or xE1-2 or xE1-3	1μM
		Wild type E2	1μM
ATP	10mM
		MgCl₂	10mM
TBS Buffer	Make up to 50. mu.L

The Western blot detection method comprises the following steps: to the reaction solution of the in vitro enzyme-linked reaction, 12.5. mu.L of 5X Loading Dye was added, and the mixture was heated at 100 ℃ for 10min to denature the protein. Electrophoresis was performed on SDS-polyacrylamide gel (in which SNS was present at a concentration of 4 wt% and polyacrylamide was present at a concentration of 15 wt%) at a voltage of 90V for concentrated gel and 120V for separation gel, in accordance with 10. mu.L per well of the denatured product of the protein. And (4) taking out the PAGE gel after electrophoresis, soaking in a Transfer buffer, and waiting for membrane Transfer. And (3) sequentially placing the PAGE glue, the NC membrane, the filter paper and the spongy cushion into a membrane transferring groove according to the principle of 'black glue and white membrane' during membrane transferring, adjusting the voltage to 110V, and transferring the membrane for 80 min. After the membrane transfer, the membrane was sealed in 5% skim milk for 1h at room temperature, and after washing the milk, primary antibody (Rabbit Anti-HA antibody) formulated with TNET was added to the membrane, which was incubated overnight in a shaker at 4 ℃. Washing the membrane with 1 XTNET solution for 10min every other day for 3 times; then adding a secondary antibody (Anti-Rabbit IgG R-800) prepared by TNET, placing the mixture in a shaking table for incubation for 1h at room temperature, and washing the membrane for 3 times by using 1x TNET solution, wherein each time lasts for 10 min. Finally, the membrane was swept using the Odyssey imaging system.

FIG. 3 is a graph showing the result of Western blot detection of mutant form xE1-1 in this example.

As can be seen from FIG. 3, in

lanes

7 and 8, there is a binding band of HA-SUMO-E1 ("-" indicates a thioester bond, the same applies hereinafter) at about 130kD, and mutant xE1-1 is reactive with both wild-type wtSUMO and mutant xSUMO5, indicating that the introduced mutation restores the activity of mutant xE1-1 and mutant xSUMO5 but fails to abolish the activity with wild-type wtSUMO.

FIG. 4 is a graph showing the result of Western blot detection of mutant form xE1-2 in this example.

As can be seen from FIG. 4, except for the HA-wtSUMO-wtE1 binding band shown in lane 5; from

lanes

7 and 8, mutant xE1-2 did not bind to the wild-type wtSUMO nor mutant xSUMO5, indicating that mutant xE1-2 lost reactivity with both wild-type wtSUMO and mutant xSUMO 5.

FIG. 5 is a graph showing the result of Western blot detection of mutant form xE1-3 in this example.

As can be seen from FIG. 5, except for the HA-wtSUMO-wtE1 binding band shown in lane 1; from

lanes

2 and 3, it can be seen that mutant xE1-3 has no binding band with both wild-type wtSUMO and mutant xSUMO5, indicating that mutant xE1-3 has lost reactivity with both wild-type wtSUMO and mutant xSUMO 5.

As can be seen from FIGS. 3, 4 and 5, only mutant xE1-1(E157R, D435R, R119E, Y159E) could restore reactivity with mutant xSUMO5, neither mutant xE1-2 nor mutant xE1-3 could react with mutant xSUMO5, and the summary of the activity assays is shown in Table 6 below. Thus, mutant xE1-1 can be used as an E1 mutant in xSUMO-xE1 combination mutants in the OST pathway; wherein, the mutant xE1-1 comprises a mutant of subunit Uba2 of SUMO activating enzyme E1, the amino acid sequence comprises the amino acid sequence shown in SEQ ID NO.5, and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 6.

Subsequent experimental studies were carried out with the mutant xE1-1 obtained in example 1.

TABLE 6

EXAMPLE 2 construction of mutant xSUMO-1, mutant xSUMO-2, and xSUMO-xE1-1 combination mutants

In this example, prokaryotic expression and purification of mutants xSUMO-1 and xSUMO-2 and mutants xSUMO-1 and xSUMO-2 were constructed, and then an xSUMO-xE1-1 combinatorial mutant was formed with mutant xE1-1 constructed in example 1, respectively, and the reaction activity of xSUMO-xE1-1 was verified by Western blot to identify a specific substrate of E2. Comprises the following steps:

1. construction of mutants of xSUMO-1 and xSUMO-2

Obtaining fragments of xSUMO-1 and xSUMO-2 by using corresponding primers through PCR, inserting a target fragment into a pET28a-HA vector digested by endonuclease in a digestion connection mode, constructing and obtaining recombinant plasmids pET28a-HA-xSUMO-1 and pET28a-HA-xSUMO-2, carrying out prokaryotic expression and purification, and determining the concentration of protein; in vitro enzyme-linked reaction was carried out, and finally, the reactivity with the mutant xE1-1 constructed in example 1 was verified by Westernblot.

1.1 PCR amplification Using the extracted cDNA as template

Mutant xSUMO5 is used as an object to introduce mutation, and the mutation principle is to change the charge type, the hydrophilic and hydrophobic properties or the molecular weight of amino acid to influence the interaction with Ubc9, so that the combination of xSUMO5 and wild-type Ubc9 in an OST pathway is eliminated. Finally, two amino acid residues of E67 and G68 are selected as mutation sites, and 2 different SUMO mutants, namely xSUMO-1 and xSUMO-2, are sequentially designed on the basis of the information of the mutation sites.

The mutation sites and primers are shown in tables 7 and 8.

TABLE 7

Name (R)	New mutation site (relative to xSUMO5)	Total mutation site (relative to wtSUMO)
			xSUMO-1	E67R，G68Y	E67R，G68Y,R70E,Y91A,E93R
xSUMO-2	G68Y	G68Y,R70E,Y91A,E93R

TABLE 8

The 50 μ L reaction system was: mu.L of xSUMO-1 or xSUMO-2, 1. mu.L of the forward primer (10. mu.M), 1. mu.L of the reverse primer (10. mu.M), 25. mu.L of rTaq Mix, 22. mu.L of ddH ₂0. Adding and uniformly mixing samples of a PCR reaction system, and putting the mixture into a PCR instrument, wherein the procedure is as follows: pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 30s, annealing at 58 deg.C for 30s, extension at 72 deg.C for 1min, reacting for 35 cycles, extension at 72 deg.C for 7min, and temporary storage at 4 deg.C.

1.2 PCR products of mutants xSUMO-1 and xSUMO-2 and plasmid pET28a-HA were digested with restriction enzymes EcoR I and XhoI, respectively, and ligated with T4 ligase

The PCR product obtained in the step 1.1) and the plasmid PET28a-HA are respectively subjected to enzyme digestion and fragment recovery by using restriction enzymes EcoR I and Xho I, and the enzyme digestion fragments and the enzyme digestion plasmids are connected to construct recombinant plasmids pET28a-HA-xSUMO-1 and pET28 a-HA-xSUMO-2.

The 30. mu.L digestion system was as follows: 200ng of the PCR product of mutant xSUMO-1 or mutant xSUMO-2 described above, 1. mu.g of PET28a-HA, 1. mu.L of EcoR I, 1. mu.L of XhoI, 3. mu.L of 10 xbuffer, plus ddH ₂0 to 30. mu.L. The reaction is carried out in a water bath at 37 ℃ for 3 h.

The 10. mu.L ligation was as follows: 7 μ L of XSUMO-1 or XSUMO-2 fragment, 1 μ L of PET28a-HA fragment, 1 μ L of 10x T4 buffer, and 1 μ L of T4 ligase.

Then respectively transforming the constructed recombinant plasmids pET28a-HA-xSUMO-1 and pET28a-HA-xSUMO-2 into DH5 alpha competent cells and coating the cells on a kana LB plate, picking 3 single clones every other day and shaking the single clones, extracting plasmids and sequencing the plasmids until xSUMO-1 and xSUMO-2 introduced into designed mutation sites are obtained, wherein the mutant xSUMO-1 comprises an amino acid sequence shown as SEQ ID NO.1, and the mutant xSUMO-1 comprises a nucleotide sequence shown as SEQ ID NO. 2; the mutant xSUMO-2 comprises an amino acid sequence shown as SEQ ID NO.3, and the mutant xSUMO-2 comprises a nucleotide sequence shown as SEQ ID NO. 4.

FIG. 6 is a 1% agarose gel electrophoresis of the resulting products of the present example, wherein mutant xSUMO-1, mutant xSUMO-2 and plasmid PET28a-HA were digested.

As can be seen from FIG. 6, the results obtained after the digestion of the PCR product and the plasmid were normal,

lanes

1 and 2 are bands of the mutant xSUMO-1, and the size is about 100 bp;

lanes

3 and 4 are bands of mutant xSUMO-2, approximately 100bp in size;

lanes

5 and 6 are bands of about 5300bp digested with plasmid PET28 a-HA.

The two recombinant plasmids pET28a-HA-xSUMO-1 and pET28a-HA-xSUMO-2 were submitted for sequencing and aligned to the template sequence using MegAlign software, the results of which are shown in FIGS. 7 and 8.

As can be seen from FIGS. 7 and 8, two mutation sites, namely E67R and G68Y, are newly introduced into mutant xSUMO-1, and one mutation site, namely G68Y, is newly introduced into mutant xSUMO-2, which indicates that both mutant xSUMO-1 and mutant xSUMO-2 are successfully constructed.

2. Prokaryotic expression and purification of mutant xSUMO-1 and mutant xSUMO-2

Respectively chemically transforming successfully-constructed recombinant plasmids pET28a-HA-xSUMO-1 and pET28a-HA-xSUMO-2 into BL21(DE3) competent cells, and performing electrophoresis on supernatant, flow-through liquid and eluent of two proteins obtained by plate coating, monoclonal picking, small shaking amplification, large shaking amplification, induced expression, high-pressure crushing, Ni-Beads affinity chromatography and centrifugal collection; the eluate was dialyzed overnight to obtain the final purified protein, and the concentration of the protein was measured. SDS-PAGE protein was electrophoresed and stained with Coomassie Brilliant blue to detect expression and purification.

FIG. 9 is an electrophoretogram of the supernatant, flow-through and eluate obtained by Ni-Beads affinity chromatography purification of mutant xSUMO-1 and mutant xSUMO-2 in this example.

As can be seen from FIG. 9,

lanes

1 and 5 show the supernatant bands of mutant xSUMO-1 and mutant xSUMO-2, and it is clear that the band of the target protein is darker around 17 KD.

Lanes

2 and 6 are the flow-through bands for mutant xSUMO-1 and mutant xSUMO-2, and the protein of interest bands were seen to be very light in color, indicating that they had bound to Ni-Beads.

Lanes

3, 4 and 7, 8 are the eluate bands of mutant xSUMO-1 and mutant xSUMO-2, and it is clear that the target bands are very dark in color and the other bands are very light in color, indicating that the eluate successfully eluted the target protein bound to Ni-Beads, and that the other proteins are few and the protein purity is high.

And dialyzing the eluent after Ni affinity chromatography purification overnight to obtain the final purified protein. The concentrations of both proteins were determined according to the instructions of the BCA kit. FIG. 15 is a standard curve obtained when the protein concentration was measured by the BCA method. The concentration of mutant xSUMO-1 was calculated to be 23. mu.M, and the concentration of mutant xSUMO-2 was calculated to be 75. mu.M. The concentration difference between mutant xSUMO-1 protein and mutant xSUMO-2 protein may be caused by the difference in the amount of elusion Buffer used during Elution, and the larger volume of eluate during Elution of mutant xSUMO-1, resulting in lower final concentration.

3. Constructing an xSUMO-xE1-1 combined mutant and verifying the reactivity by Western blot

FIG. 10 is a Western blot analysis result of the mutant xSUMO5-xE1-1 combination in this example.

As can be seen from FIG. 10, mutant xSUMO5 did not bind to wild-type wtE1, but could bind to mutant xE1-1, demonstrating the successful construction of a combination mutant of xSUMO5-xE 1-1.

FIG. 11 is a Western blot analysis result of wild-type E2 added to the mutant xSUMO5-xE1-1 in this example.

As can be seen from fig. 11, mutant xSUMO5 was still able to be delivered to wild-type E2 via wild-type wtE 1. The reason is that: from FIG. 10, it can be seen that mutant xSUMO5 binds weakly to wild-type wtE1, but still has very weak binding ability, and the addition of wild-type E2 to xSUMO5-wtE1-1 amplifies the phenomenon of small amount of mutant xSUMO5 transmission in wild-type wtE1 due to the polysufylation reaction of wild-type E2.

FIG. 12 is a graph showing the results of Western blot analysis of mutant XSUMO-1 and mutant XSUMO-2 in this example.

As can be seen from fig. 12,

lanes

1 and 2 are the activity verification of wild-type wtSUMO, and the band at 130KD in lane 1 is the band where wtSUMO and wtE1 are bound; the diffuse band in lane 2 is the polysumo reaction band where wtSUMO was delivered via wtE1 to wtE2, which demonstrates that all wild-type proteins used are reactive. Lanes 3 to 7 are the activity verification of mutant xSUMO-1, and the band at 15KD of lane 3 is xSUMO-1, which proves that the HA tag is successfully expressed together with the target protein; lane 4 is the reaction of mutant xSUMO-1 with wild-type wtE1, with no band at 130KD, demonstrating its lack of binding activity to wild-type wtE 1; lane 5 is the reaction of mutant xSUMO-1 with mutant xE1-1, a 130KD band demonstrating the reactivity of both; lane 6 has no band, demonstrating that mutant xSUMO-1 is not delivered to wild-type E2 via wild-type wtE 1; the diffuse band in lane 7 is the polysufo reaction band where mutant xSUMO-1 binds to wild-type E2 via mutant xE1-1, demonstrating that xSUMO-1 is reactive with mutant xE1-1 and can be normally transferred to wild-type E2, and that wild-type wtSUMO and wild-type wtE1 do not interfere with the mutation pathway. Lanes 8 to 12 demonstrate the activity of mutant xSUMO-2, which is very similar to mutant xSUMO-1, but the diffuse band in lane 12 is very faint, demonstrating that mutant xSUMO-2 has a weak binding activity to wild-type E2.

FIG. 13 shows the addition of a RanGTP enzyme agonist protein, namely RanGAP, which is a specific substrate for E2 in a hybridization validation experiment.

From FIG. 13,

lanes

1, 2, 4, 5, again demonstrate that mutant xSUMO-1 and mutant xSUMO-2 are not transmitted to wild-type E2 via the wild-type wtE1 pathway, but are transmitted to wild-type E2 via mutant xE 1-1. The results in lane 3 and lane 6 show that mutant xSUMO-1 and mutant xSUMO-2 can be transferred to wild-type E2 through mutant xE1-1, and then transferred to E2 specific substrate RanGAP from wild-type E2, which indicates that mutant xSUMO-1 and mutant xSUMO-2 respectively form qualified combined mutants with mutant xE1-1, and xSUMO-1-xE1-1 and xSUMO-2-xE1-1 both have activity of transferring to substrate RanGAP, but are not affected by wild-type wtE1 or wild-type wtSUMO, and have strong specificity, and can be used for identifying specific substrates of Ubc9 and specific substrates of E3.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Sequence listing

<110> Shanghai university of transportation

<120> mutant xSUMO, mutant xE1 and xSUMO-xE1 combination mutants and related products and uses thereof

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 97

<212> PRT

<213> SEQ ID：1(Artificial Sequence)

<400> 1

Met Ser Asp Gln Glu Ala Lys Pro Ser Thr Glu Asp Leu Gly Asp Lys

1 5 10 15

Lys Glu Gly Glu Tyr Ile Lys Leu Lys Val Ile Gly Gln Asp Ser Ser

20 25 30

Glu Ile His Phe Lys Val Lys Met Thr Thr His Leu Lys Lys Leu Lys

35 40 45

Glu Ser Tyr Cys Gln Arg Gln Gly Val Pro Met Asn Ser Leu Arg Phe

50 55 60

Leu Phe Arg Tyr Gln Glu Ile Ala Asp Asn His Thr Pro Lys Glu Leu

65 70 75 80

Gly Met Glu Glu Glu Asp Val Ile Glu Val Ala Gln Arg Gln Thr Gly

85 90 95

Gly

<210> 2

<211> 294

<212> DNA

<213> SEQ ID：2(Artificial Sequence)

<400> 2

atgtctgacc aggaggcaaa accttcaact gaggacttgg gggataagaa ggaaggtgaa 60

tatattaaac tcaaagtcat tggacaggat agcagtgaga ttcacttcaa agtgaaaatg 120

acaacacatc tcaagaaact caaagaatca tactgtcaaa gacagggtgt tccaatgaat 180

tcactcaggt ttctctttag gtatcaggaa attgctgata atcatactcc aaaagaactg 240

ggaatggagg aagaagatgt gattgaagtt gctcagagac aaacgggggg ttag 294

<210> 3

<211> 97

<212> PRT

<213> SEQ ID：3(Artificial Sequence)

<400> 3

Met Ser Asp Gln Glu Ala Lys Pro Ser Thr Glu Asp Leu Gly Asp Lys

1 5 10 15

Lys Glu Gly Glu Tyr Ile Lys Leu Lys Val Ile Gly Gln Asp Ser Ser

20 25 30

Glu Ile His Phe Lys Val Lys Met Thr Thr His Leu Lys Lys Leu Lys

35 40 45

Glu Ser Tyr Cys Gln Arg Gln Gly Val Pro Met Asn Ser Leu Arg Phe

50 55 60

Leu Phe Glu Tyr Gln Glu Ile Ala Asp Asn His Thr Pro Lys Glu Leu

65 70 75 80

Gly Met Glu Glu Glu Asp Val Ile Glu Val Ala Gln Arg Gln Thr Gly

85 90 95

Gly

<210> 4

<211> 294

<212> DNA

<213> SEQ ID：4(Artificial Sequence)

<400> 4

atgtctgacc aggaggcaaa accttcaact gaggacttgg gggataagaa ggaaggtgaa 60

tatattaaac tcaaagtcat tggacaggat agcagtgaga ttcacttcaa agtgaaaatg 120

acaacacatc tcaagaaact caaagaatca tactgtcaaa gacagggtgt tccaatgaat 180

tcactcaggt ttctctttga gtatcaggaa attgctgata atcatactcc aaaagaactg 240

ggaatggagg aagaagatgt gattgaagtt gctcagagac aaacgggggg ttag 294

<210> 5

<211> 640

<212> PRT

<213> SEQ ID：5(Artificial Sequence)

<400> 5

Met Ala Leu Ser Arg Gly Leu Pro Arg Glu Leu Ala Glu Ala Val Ala

1 5 10 15

Gly Gly Arg Val Leu Val Val Gly Ala Gly Gly Ile Gly Cys Glu Leu

20 25 30

Leu Lys Asn Leu Val Leu Thr Gly Phe Ser His Ile Asp Leu Ile Asp

35 40 45

Leu Asp Thr Ile Asp Val Ser Asn Leu Asn Arg Gln Phe Leu Phe Gln

50 55 60

Lys Lys His Val Gly Arg Ser Lys Ala Gln Val Ala Lys Glu Ser Val

65 70 75 80

Leu Gln Phe Tyr Pro Lys Ala Asn Ile Val Ala Tyr His Asp Ser Ile

85 90 95

Met Asn Pro Asp Tyr Asn Val Glu Phe Phe Arg Gln Phe Ile Leu Val

100 105 110

Met Asn Ala Leu Asp Asn Glu Ala Ala Arg Asn His Val Asn Arg Met

115 120 125

Cys Leu Ala Ala Asp Val Pro Leu Ile Glu Ser Gly Thr Ala Gly Tyr

130 135 140

Leu Gly Gln Val Thr Thr Ile Lys Lys Gly Val Thr Arg Cys Glu Glu

145 150 155 160

Cys His Pro Lys Pro Thr Gln Arg Thr Phe Pro Gly Cys Thr Ile Arg

165 170 175

Asn Thr Pro Ser Glu Pro Ile His Cys Ile Val Trp Ala Lys Tyr Leu

180 185 190

Phe Asn Gln Leu Phe Gly Glu Glu Asp Ala Asp Gln Glu Val Ser Pro

195 200 205

Asp Arg Ala Asp Pro Glu Ala Ala Trp Glu Pro Thr Glu Ala Glu Ala

210 215 220

Arg Ala Arg Ala Ser Asn Glu Asp Gly Asp Ile Lys Arg Ile Ser Thr

225 230 235 240

Lys Glu Trp Ala Lys Ser Thr Gly Tyr Asp Pro Val Lys Leu Phe Thr

245 250 255

Lys Leu Phe Lys Asp Asp Ile Arg Tyr Leu Leu Thr Met Asp Lys Leu

260 265 270

Trp Arg Lys Arg Lys Pro Pro Val Pro Leu Asp Trp Ala Glu Val Gln

275 280 285

Ser Gln Gly Glu Glu Thr Asn Ala Ser Asp Gln Gln Asn Glu Pro Gln

290 295 300

Leu Gly Leu Lys Asp Gln Gln Val Leu Asp Val Lys Ser Tyr Ala Arg

305 310 315 320

Leu Phe Ser Lys Ser Ile Glu Thr Leu Arg Val His Leu Ala Glu Lys

325 330 335

Gly Asp Gly Ala Glu Leu Ile Trp Asp Lys Asp Asp Pro Ser Ala Met

340 345 350

Asp Phe Val Thr Ser Ala Ala Asn Leu Arg Met His Ile Phe Ser Met

355 360 365

Asn Met Lys Ser Arg Phe Asp Ile Lys Ser Met Ala Gly Asn Ile Ile

370 375 380

Pro Ala Ile Ala Thr Thr Asn Ala Val Ile Ala Gly Leu Ile Val Leu

385 390 395 400

Glu Gly Leu Lys Ile Leu Ser Gly Lys Ile Asp Gln Cys Arg Thr Ile

405 410 415

Phe Leu Asn Lys Gln Pro Asn Pro Arg Lys Lys Leu Leu Val Pro Cys

420 425 430

Ala Leu Arg Pro Pro Asn Pro Asn Cys Tyr Val Cys Ala Ser Lys Pro

435 440 445

Glu Val Thr Val Arg Leu Asn Val His Lys Val Thr Val Leu Thr Leu

450 455 460

Gln Asp Lys Ile Val Lys Glu Lys Phe Ala Met Val Ala Pro Asp Val

465 470 475 480

Gln Ile Glu Asp Gly Lys Gly Thr Ile Leu Ile Ser Ser Glu Glu Gly

485 490 495

Glu Thr Glu Ala Asn Asn His Lys Lys Leu Ser Glu Phe Gly Ile Arg

500 505 510

Asn Gly Ser Arg Leu Gln Ala Asp Asp Phe Leu Gln Asp Tyr Thr Leu

515 520 525

Leu Ile Asn Ile Leu His Ser Glu Asp Leu Gly Lys Asp Val Glu Phe

530 535 540

Glu Val Val Gly Asp Ala Pro Glu Lys Val Gly Pro Lys Gln Ala Glu

545 550 555 560

Asp Ala Ala Lys Ser Ile Thr Asn Gly Ser Asp Asp Gly Ala Gln Pro

565 570 575

Ser Thr Ser Thr Ala Gln Glu Gln Asp Asp Val Leu Ile Val Asp Ser

580 585 590

Asp Glu Glu Asp Ser Ser Asn Asn Ala Asp Val Ser Glu Glu Glu Arg

595 600 605

Ser Arg Lys Arg Lys Leu Asp Glu Lys Glu Asn Leu Ser Ala Lys Arg

610 615 620

Ser Arg Ile Glu Gln Lys Glu Glu Leu Asp Asp Val Ile Ala Leu Asp

625 630 635 640

<210> 6

<211> 1923

<212> DNA

<213> SEQ ID：6(Artificial Sequence)

<400> 6

atggcactgt cgcgggggct gccccgggag ctggctgagg cggtggccgg gggccgggtg 60

ctggtggtgg gggcgggcgg catcggctgc gagctcctca agaatctcgt gctcaccggt 120

ttctcccaca tcgacctgat tgatctggat actattgatg taagcaacct caacagacag 180

tttttgtttc aaaagaaaca tgttggaaga tcaaaggcac aggttgccaa ggaaagtgta 240

ctgcagtttt acccgaaagc taatatcgtt gcctaccatg acagcatcat gaaccctgac 300

tataatgtgg aatttttccg acagtttata ctggttatga atgctttaga taacgaagct 360

gcccgaaacc atgttaatag aatgtgcctg gcagctgatg ttcctcttat tgaaagtgga 420

acagctgggt atcttggaca agtaactact atcaaaaagg gtgtgacccg gtgtgaggag 480

tgtcatccta agccgaccca gagaaccttt cctggctgta caattcgtaa cacaccttca 540

gaacctatac attgcatcgt ttgggcaaag tacttgttca accagttgtt tggggaagaa 600

gatgctgatc aagaagtatc tcctgacaga gctgaccctg aagctgcctg ggaaccaacg 660

gaagccgaag ccagagctag agcatctaat gaagatggtg acattaaacg tatttctact 720

aaggaatggg ctaaatcaac tggatatgat ccagttaaac tttttaccaa gctttttaaa 780

gatgacatca ggtatctgtt gacaatggac aaactatggc ggaaaaggaa acctccagtt 840

ccgttggact gggctgaagt acaaagtcaa ggagaagaaa cgaatgcatc agatcaacag 900

aatgaacccc agttaggcct gaaagaccag caggttctag atgtaaagag ctatgcacgt 960

cttttttcaa agagcatcga gactttgaga gttcatttag cagaaaaggg ggatggagct 1020

gagctcatat gggataagga tgacccatct gcaatggatt ttgtcacctc tgctgcaaac 1080

ctcaggatgc atattttcag tatgaatatg aagagtagat ttgatatcaa atcaatggca 1140

gggaacatta ttcctgctat tgctactact aatgcagtaa ttgctgggtt gatagtattg 1200

gaaggattga agattttatc aggaaaaata gaccagtgca gaacaatttt tttgaataaa 1260

caaccaaacc caagaaagaa gcttcttgtg ccttgtgcac tgcgtcctcc caaccccaat 1320

tgttatgtat gtgccagcaa gccagaggtg actgtgcggc tgaatgtcca taaagtgact 1380

gttctcacct tacaagacaa gatagtgaaa gaaaaatttg ctatggtagc accagatgtc 1440

caaattgaag atgggaaagg aacaatccta atatcttccg aagagggaga gacggaagct 1500

aataatcaca agaagttgtc agaatttgga attagaaatg gcagccggct tcaagcagat 1560

gacttcctcc aggactatac tttattgatc aacatccttc atagtgaaga cctaggaaag 1620

gacgttgaat ttgaagttgt tggtgatgcc ccggaaaaag tggggcccaa acaagctgaa 1680

gatgctgcca aaagcataac caatggcagt gatgatggag ctcagccctc cacctccaca 1740

gctcaagagc aagatgacgt tctcatagtt gattcggatg aagaagattc ttcaaataat 1800

gccgacgtca gtgaagaaga gagaagccgc aagaggaaat tagatgagaa agagaatctc 1860

agtgcaaaga ggtcacgtat agaacagaag gaagagcttg atgatgtcat agcattagat 1920

taa 1923

<210> 7

<211> 294

<212> DNA

<213> SEQ ID：7(Artificial Sequence)

<400> 7

atgtctgacc aggaggcaaa accttcaact gaggacttgg gggataagaa ggaaggtgaa 60

tatattaaac tcaaagtcat tggacaggat agcagtgaga ttcacttcaa agtgaaaatg 120

acaacacatc tcaagaaact caaagaatca tactgtcaaa gacagggtgt tccaatgaat 180

tcactcaggt ttctctttga gggtcaggaa attgctgata atcatactcc aaaagaactg 240

ggaatggagg aagaagatgt gattgaagtt gctcagagac aaacgggggg ttag 294

<210> 8

<211> 346

<212> PRT

<213> SEQ ID：8(Artificial Sequence)

<400> 8

Met Val Glu Lys Glu Glu Ala Gly Gly Gly Ile Ser Glu Glu Glu Ala

1 5 10 15

Ala Gln Tyr Asp Arg Gln Ile Arg Leu Trp Gly Leu Glu Ala Gln Lys

20 25 30

Arg Leu Arg Ala Ser Arg Val Leu Leu Val Gly Leu Lys Gly Leu Gly

35 40 45

Ala Glu Ile Ala Lys Asn Leu Ile Leu Ala Gly Val Lys Gly Leu Thr

50 55 60

Met Leu Asp His Glu Gln Val Thr Pro Glu Asp Pro Gly Ala Gln Phe

65 70 75 80

Leu Ile Arg Thr Gly Ser Val Gly Arg Asn Arg Ala Glu Ala Ser Leu

85 90 95

Glu Arg Ala Gln Asn Leu Asn Pro Met Val Asp Val Lys Val Asp Thr

100 105 110

Glu Asp Ile Glu Lys Lys Pro Glu Ser Phe Phe Thr Gln Phe Asp Ala

115 120 125

Val Cys Leu Thr Cys Cys Ser Arg Asp Val Ile Val Lys Val Asp Gln

130 135 140

Ile Cys His Lys Asn Ser Ile Lys Phe Phe Thr Gly Asp Val Phe Gly

145 150 155 160

Tyr His Gly Tyr Thr Phe Ala Asn Leu Gly Glu His Glu Phe Val Glu

165 170 175

Glu Lys Thr Lys Val Ala Lys Val Ser Gln Gly Val Glu Asp Gly Pro

180 185 190

Asp Thr Lys Arg Ala Lys Leu Asp Ser Ser Glu Thr Thr Met Val Lys

195 200 205

Lys Lys Val Val Phe Cys Pro Val Lys Glu Ala Leu Glu Val Asp Trp

210 215 220

Ser Ser Glu Lys Ala Lys Ala Ala Leu Lys Arg Thr Thr Ser Asp Tyr

225 230 235 240

Phe Leu Leu Gln Val Leu Leu Lys Phe Arg Thr Asp Lys Gly Arg Asp

245 250 255

Pro Ser Ser Asp Thr Tyr Glu Glu Asp Ser Glu Leu Leu Leu Gln Ile

260 265 270

Arg Asn Asp Val Leu Asp Ser Leu Gly Ile Ser Pro Asp Leu Leu Pro

275 280 285

Glu Asp Phe Val Arg Tyr Cys Phe Ser Glu Met Ala Pro Val Cys Ala

290 295 300

Val Val Gly Gly Ile Leu Ala Gln Glu Ile Val Lys Ala Leu Ser Gln

305 310 315 320

Arg Asp Pro Pro His Asn Asn Phe Phe Phe Phe Asp Gly Met Lys Gly

325 330 335

Asn Gly Ile Val Glu Cys Leu Gly Pro Lys

340 345

<210> 9

<211> 1041

<212> DNA

<213> SEQ ID：9(Artificial Sequence)

<400> 9

atggtggaga aggaggaggc tggcggcggc attagcgagg aggaggcggc acagtatgac 60

cggcagatcc gcctgtgggg actggaggcc cagaaacggc tgcgggcctc tcgggtgctt 120

cttgtcggct tgaaaggact tggggctgaa attgccaaga atctcatctt ggcaggagtg 180

aaaggactga ccatgctgga tcacgaacag gtaactccag aagatcccgg agctcagttc 240

ttgattcgta ctgggtctgt tggccgaaat agggctgaag cctctttgga gcgagctcag 300

aatctcaacc ccatggtgga tgtgaaggtg gacactgagg atatagagaa gaaaccagag 360

tcatttttca ctcaattcga tgctgtgtgt ctgacttgct gctccaggga tgtcatagtt 420

aaagttgacc agatctgtca caaaaatagc atcaagttct ttacaggaga tgtttttggc 480

taccatggat acacatttgc caatctagga gagcatgagt ttgtagagga gaaaactaaa 540

gttgccaaag ttagccaagg agtagaagat gggcccgaca ccaagagagc aaaacttgat 600

tcttctgaga caacgatggt caaaaagaag gtggtcttct gccctgttaa agaagccctg 660

gaggtggact ggagcagtga gaaagcaaag gctgctctga agcgcacgac ctccgactac 720

tttctccttc aagtgctctt aaagttccgt acagataaag gaagagatcc cagttctgat 780

acatatgagg aagattctga gttgttgctc cagatacgaa atgatgtgct tgactcactg 840

ggtattagtc ctgacctgct tcctgaggac tttgtcaggt actgcttctc cgagatggcc 900

ccagtgtgtg cggtggttgg agggattttg gcacaggaaa ttgtgaaggc cctgtctcag 960

cgggaccctc ctcacaacaa cttcttcttc ttcgatggca tgaaggggaa tgggattgtg 1020

gagtgccttg gccccaagtg a 1041

Claims

1. The mutant xSUMO is characterized in that the mutant is the protein as shown in the following (1) or (2):

2. The biological material associated with the mutant xSUMO of claim 1, comprising any one of:

a) a polynucleotide encoding the mutant xSUMO of claim 1;

b) a recombinant expression vector comprising the polynucleotide of a);

3. The biomaterial of claim 2, wherein in a) the polynucleotide comprises a nucleotide sequence as set forth in SEQ ID No.2 or SEQ ID No. 4.

4. The mutant xE1 is characterized in that the mutant is a SUMO activating enzyme E1 subunit Uba2 mutant, and the mutant is a protein as shown in the following (1) or (2):

(1) a protein consisting of an amino acid sequence shown as SEQ ID No. 5;

5. Biological material related to the mutant xE1 of claim 4, comprising any of the following:

a) a polynucleotide encoding the mutant xE1 of claim 4;

b) a recombinant expression vector comprising the polynucleotide of a);

6. The biomaterial of claim 5, wherein in a) the polynucleotide comprises a nucleotide sequence as set forth in SEQ ID No. 6.

An xSUMO-xE1 combination mutant, wherein said xSUMO-xE1 combination mutant comprises the mutant xSUMO of claim 1;

and/or, the xSUMO-xE1 combination mutant comprises mutant xE1 of claim 4.

8. Biomaterial associated with the xSUMO-xE1 combination mutant according to claim 7, comprising any of the following:

a) polynucleotides encoding the xSUMO-xE1 combination mutant of claim 7;

b) a recombinant expression vector comprising the polynucleotide of a);

9. Use of the mutant xSUMO of claim 1 or the biomaterial related to mutant xSUMO of claim 2 or 3 or the mutant xE1 of claim 4 or the biomaterial related to mutant xE1 of claim 5 or 6 or the xSUMO-xE1 combination mutant of claim 7 or the biomaterial related to xSUMO-xE1 combination mutant of claim 8 for identifying a Ubc9 substrate or an E3 substrate.

10. Use of the mutant xSUMO of claim 1 or the biomaterial related to mutant xSUMO of claim 2 or 3 or the mutant xE1 of claim 4 or the biomaterial related to mutant xE1 of claim 5 or 6 or the xSUMO-xE1 combination mutant of claim 7 or the biomaterial related to xSUMO-xE1 combination mutant of claim 8 for screening anti-neurodegenerative diseases or anti-cancer drugs.