Method for efficiently obtaining Cullin1-Rbx1 E3 ligase complex by prokaryotic expression

Abstract

The invention belongs to the technical field of protein purification and relates to a method for efficiently obtaining a Cullin1-Rbx1 E3 ligase complex by prokaryotic expression. The method comprises reacting an optimized and modified Cullin1-Rbx1 fusion protein with Skp1-Skp2 and Cks1 in a reaction system to obtain the E3 ligase complex. Introducing an MsyB tag at the N-terminus of Cullin1 can improve the solubility of the protein and solve the problem that Cullin1 is difficult to fold in a prokaryotic expression system. Cullin1 and Rbx1 are co-expressed to reduce misfolding and aggregation when each exists alone. These improvements enable the Cullin1-RBX1 fusion protein to be conveniently and efficiently expressed and purified in a shorter time, laying a foundation for subsequent biochemical structure mechanism research. The method for preparing the E3 ligase complex provided by the invention is simple and efficient and is suitable for large-scale promotion and use.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein purification and relates to a method for efficiently obtaining a Cullin1-Rbx1 E3 ligase complex through prokaryotic expression. Background Art

[0002] Ubiquitination is the process of covalently attaching small molecule protein ubiquitin to substrate protein through three enzyme cascade reactions: E1, E2 and E3. The process involves E1 enzyme activating ubiquitin, E2 enzyme transferring ubiquitin, and E3 enzyme recognizing substrate and attaching ubiquitin to substrate. Ubiquitination can not only attach alone, but also form different types of polyubiquitin chains through the seven lysine residues (K6, K11, K27, K29, K33, K48, K63) or N-terminal methionine (M1) of ubiquitin, regulating protein degradation, activity and intracellular localization and other functions. Cullin1-RING E3 ligase complex (CRL) is the largest and most diverse class in the E3 ubiquitin ligase family, composed of scaffold protein Cullin1, RING protein (such as RBX1), adapter protein and substrate recognition protein. Cullin1 protein acts as a scaffold to ensure the smooth transfer of ubiquitin from E2 enzyme to substrate protein through interaction with RING protein and substrate recognition module. CRL1 plays a key role in biological processes such as cell cycle regulation, DNA repair, and signal transduction. Its disorder is closely related to many diseases, especially cancer. In the ubiquitination process catalyzed by Cullin1, the K48-linked polyubiquitin chain is a classic example. First, the E2 enzyme Ube2D links the first ubiquitin molecule to the substrate protein, and then the ubiquitin chain is extended through the K48 linking site under the action of the E2 enzyme Cdc34. The formation of the K48 chain is a protein degradation signal, which is recognized and degraded by the proteasome, thereby regulating the cell cycle and gene expression. As the core component of the CRL complex, the catalytic mechanism of Cullin1 is crucial to understanding the biochemical mechanism of the ubiquitination process. The study of CRL not only reveals the molecular mechanism of ubiquitination, but also provides potential drug targets for disease treatment.

[0003] However, efficient expression and purification of Cullin1-Rbx1 has always been a bottleneck problem in structural biology research. Traditionally, researchers mainly use insect cell expression systems to achieve the expression of Cullin1-Rbx1. However, the insect cell expression system also has significant limitations. First, its expression cycle is long, and it usually takes one month from viral transformation to protein harvesting, which greatly reduces the efficiency of the experiment. Secondly, the cost of insect cell culture and baculovirus production is high, which increases the economic burden of the experiment. In addition, the operation is more complicated and requires strict experimental conditions and professional technicians, which to some extent limits its wide application. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for efficiently and conveniently preparing the Cullin1-Rbx1 E3 enzyme complex in vitro, wherein the optimized Cullin1-Rbx1 fusion protein is reacted with Skp1-Skp2 and Cks1 in a reaction system to obtain the E3 ligase complex.

[0005] In order to achieve the above object, the present invention provides a method for efficiently obtaining a Cullin1-Rbx1 E3 ligase complex by prokaryotic expression, wherein the structures of the Cullin1 gene and the Rbx1 gene are firstly optimized respectively, and Cullin1 and Rbx1 are co-expressed to obtain a Cullin1-Rbx1 fusion protein, and then the Cullin1-Rbx1 fusion protein is reacted with Skp1-Skp2 and Cks1 in a reaction system to obtain a Cullin1-Rbx1 E3 ligase complex; the specific steps are as follows:

[0006] (1) Obtaining Cullin1-Rbx1 fusion protein by prokaryotic expression:

[0007] 1) Construction of the recombinant vector MsyB-Cullin1-Rbx1-His: In order to construct a vector for the simultaneous expression of Cullin1 and RBX1, the full-length human Cullin1 gene was first codon-optimized, and the MsyB solubilization tag and HRV3C restriction site were added to its N-terminus. Then, the optimized Cullin1 was cloned behind the first ribosome binding site (RBS) of the pET29a vector; Next, the full-length human Rbx1 gene was codon-optimized, and a 6X His tag and HRV 3C restriction site were added to its N-terminus. It was then cloned behind the second ribosome binding site (RBS) of the pET29a vector to obtain the recombinant vector MsyB-Cullin1-Rbx1-His;

[0008] 2) Expression of fusion protein Cullin1-Rbx1: The obtained recombinant vector MsyB-Cullin1-Rbx1-His was transformed into Escherichia coli BL21 (DE3) by heat shock method and cultured overnight on LB plates containing kanamycin at a final concentration of 50 μg / ml. The colonies grown after overnight culture were regarded as positive colonies.

[0009] Take a positive single colony containing the recombinant plasmid and inoculate it into LB liquid medium containing a final concentration of 50 μg / ml kanamycin. Incubate it at 37°C and 200 rpm for 10 to 12 hours. Inoculate the cultured bacterial liquid into fresh LB liquid medium containing 50 μg / ml kanamycin at a volume ratio of 1:50. Incubate it at 37°C and 200 rpm until the bacterial liquid concentration OD600 is 0.8 to 1.2. Then add the inducer IPTG (isopropyl-β-D-thiogalactopyranoside) to a working concentration of 0.2 mM. Continue to culture at 16°C and 180 rpm for 10-26 hours, centrifuge for 20 minutes, collect and resuspend the bacteria.

[0010] The collected bacteria were resuspended in 50mM HEPES 150mM NaCl buffer (pH=7.5), PMSF (phenylmethylsulfonyl fluoride) was added to a working concentration of 1mM, mixed, and ultrasonically disrupted on ice. The ultrasonic parameters were as follows: power 100W, ultrasonication 3s, interval 6s, a total of 40min; then centrifuged at 4°C, 12000rpm for 30min to obtain the supernatant containing protein;

[0011] 3) Purification of fusion protein Cullin1-Rbx1: Purification was performed using a nickel column. The supernatant was incubated with the nickel column and then washed and eluted with 20mM and 300mM imidazole. The eluate was collected and the protein was cleaved with PreScission protease to remove the His tag. The eluate was dialyzed overnight in dialysis buffer (50 mM MES, 50 mM NaCl, pH 6.5). The next day, the sample was filtered with a 0.22μm filter membrane and loaded onto a Source S 10 / 300GL column pre-equilibrated with buffer (50mM MES, pH 6.5). Linear gradient elution was performed to separate the purified Cullin1-Rbx1 fusion protein. Using this method, 2 mg of protein could be purified per liter of bacteria.

[0012] (2) Preparation of Cullin1-Rbx1 E3 ligase:

[0013] The protein components, adaptor proteins Skp1-Skp2, Cks1 and Cullin1-RBX1, were reacted in a reaction system under the following reaction conditions: incubation on ice for one hour to obtain a Cullin1-Rbx1 E3 ligase complex.

[0014] The reaction system also contains 50 mM HEPES 150 mM NaCl buffer (pH 7.5), and the contents of various substances in the system are: 1 μM Skp1-Skp2, 1 μM Cks1 and 1 μM Cullin1-Rbx1.

[0015] Compared with the prior art, the present invention efficiently and conveniently prepares the Cullin1-Rbx1E3 enzyme complex in vitro using the modified Cullin1-Rbx1E3 fusion protein; introducing the MsyB tag at the N-terminus of Cullin1 can improve the solubility of the protein and solve the problem that Cullin1 is difficult to fold in the prokaryotic expression system; in addition, by co-expressing Cullin1 and Rbx1, Cullin1 and RBX1 form a stable complex, reducing the misfolding and aggregation when each exists alone; these improvements enable the Cullin1-RBX1 fusion protein to be conveniently and efficiently expressed and purified in a shorter time, laying a foundation for subsequent biochemical structural mechanism research; compared with the insect cell expression system which takes nearly one month, the advantage of the method of the present invention is that 2 mg of Cullin1-Rbx1 fusion protein can be conveniently obtained per liter of bacteria within one week, which fully meets the requirements of corresponding biochemical characteristics, structural mechanisms and active molecular screening; the method of the present invention for preparing the Cullin1-Rbx1 E3 ligase complex is simple and efficient, and is suitable for large-scale promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the structure of the Cullin1-Rbx1 fusion protein plasmid in Example 1 of the present invention.

[0017] Figure 2 This is the SDS-PAGE electrophoresis diagram of the Cullin1-Rbx1 fusion protein induced to be expressed in Example 1 of the present invention and purified by nickel column and Source S 10 / 300GL chromatographic column, wherein A is the electrophoresis diagram after purification by nickel column; B is the electrophoresis diagram after removal of His tag; C is the electrophoresis diagram after purification by Source S 10 / 300GL chromatographic column; M is the protein molecular weight standard.

[0018] Figure 3 This is an SDS PAGE electrophoresis diagram of Example 2 of the present invention for verifying whether the fusion protein Cullin1-Rbx1 has activity in an in vitro ubiquitination-like modification system, wherein A is a schematic diagram of the reaction process and B is an electrophoresis diagram.

[0019] Figure 4 This is an SDS PAGE electrophoresis diagram for verifying whether the fusion protein Cullin1-Rbx1 self-assembles into an E3 enzyme complex in vitro in Example 3 of the present invention. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described below through specific embodiments in conjunction with the accompanying drawings.

[0021] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0022] The preparation method of 50mM HEPES 150mM NaCl buffer (pH 7.5) in the embodiment is: HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)) and NaCl are dissolved in water, and the pH is adjusted to 7.5 with NaOH, wherein the final concentration of HEPES is 50mM, and the final concentration of NaCl is 150mM.

[0023] Example 1: Prokaryotic expression of in vitro recombinant RING E3 ligase activity and its application

[0024] In order to simplify the in vitro activity reconstitution system of multi-subunit RING E3 ligase, this example fused Cullin1 and RBX1 into a fusion protein ( Figure 1 ).

[0025] 1. Preparation of recombinant vector

[0026] In order to construct a vector for simultaneously expressing Cullin1 and RBX1, the present embodiment uses a pET29a vector, the promoter of which is a T7 promoter; the specific steps are as follows: first, the full-length human Cullin1 gene is codon-optimized, and in order to increase the solubility of Cullin1, an MsyB solubility-promoting tag and an HRV 3C restriction site are added to its N-terminus, and the MsyB tag and Cullin1 are separated by the HRV 3C restriction site. Then, the optimized Cullin1 is cloned behind the first ribosome binding site (RBS) of the pET29a vector (the cloning process is completed by Nanjing GenScript Biotechnology Co., Ltd.; the N-terminus of Cullin1 is connected to the 1430th base pair of the pET29a vector, and the C-terminus is terminated at the 3856th base pair); for the full-length human RBX1 gene, codon optimization is also performed, and a 6X His tag and an HRV 3C restriction site are added to its N-terminus, and the 6X His tag and RBX1 are separated by the HRV The 3C restriction site was separated and then cloned into the second ribosome binding site (RBS) of the pET29a vector (the cloning process was completed by Nanjing GenScript Biotechnology Co., Ltd.); finally, the obtained recombinant vector was recorded as MsyB-Cullin1-RBX1-His;

[0027] The DNA sequence of the Cullin1 gene is as follows:

[0028]

[0029] The amino acid sequence encoded by the Cullin1 gene is as follows:

[0030] MSSTRSQNPHGLKQIGLDQIWDDLRAGIQQVYTRQSMAKSRYMELYTHVYNYCTSVHQSNQARGAGVPPSKSKKGQTPGGAQFVGLELYKRLKEFLKNYLTNLLKDGEDLMDESVLKFYTQQWEDYRFSSKVLNGICAYLNRHWVRRECDEGRKGIYEIYSLALVTWRDCLFRPLNKQVTNAVLKLIEKERNGETINTRLISGVVQSYVELGLNEDDAFAKGPTLTVYKESFESQFLADTERFYTRESTEFLQQNPVTEYMKKAEARLLEEQRRVQVYLHESTQDELARKCEQVLIEKHLEIFHTEFQNLLDADKNEDLGRMYNLVSRIQDGLGELKKLLETHIHNQGLAAIEKCGEAALNDPKMYVQTVLDVHKKYNALVMSAFNNDAGFVAALDKACGRFINNNAVTKMAQSSSKSPELLARYCDSLLKKSSKNPEEAELEDTLNQVMVVFKYIEDKDVFQKFYAKMLAKRLVHQNSASDDAEASMISKLKQACGFEYTSKLQRMFQDIGVSKDLNEQFKKHLTNSEPLDLDFSIQVLSSGSWPFQQSCTFALPSELERSYQRFTAFYASRHSGRKLTWLYQLSKGELVTNCFKNRYTLQASTFQMAILLQYNTEDAYTVQQLTDSTQIKMDILAQVLQILLKSKLLVLEDENANVDEVELKPDTLIKLYLGYKNKKLRVNINVPMKTEQKQEQETTHKNIEEDRKLLIQAAIVRIMKMRKVLKHQQLLGEVLTQLSSRFKPRVPVIKKCIDILIEKEYLERVDGEKDTYSYLA (SEQ ID NO:2).

[0031] The DNA sequence of the RBX1 gene is as follows:

[0032] Cgctttgaagtgaagaagtggaatgcagttgctctgtgggcatgggatattgttgttgacaactgcgcgatttgccgtaatcacattatggacctttgcatcgaatgtcaggctaatcaggcgagcgcgaccagc gaagagtgcaccgttgcgtggggtgtgtgcaaccacgccttccactttcattgtatttctcgttggctgaagacccgtcaagtctgtccgctggacaaccgcgagtgggagttccagaaatatggtcattaa (seq ID NO:3).

[0033] The amino acid sequence encoded by the RBX1 gene is as follows:

[0034] RFEVKKWNAVALWAWDIVVDNCAICRNHIMDLCIECQANQASATSEECTVAWGVCNHAFHFHCISRWLKTRQVCPLDNREWEFQKYGH (SEQ ID NO: 4).

[0035] The amino acid sequence of the MsyB is as follows:

[0036] MTMYATLEEAIDAAREEFLADNPGIDAEDANVQQFNAQKYVLQDGDIMWQVEFFADEGEEGECLPMLSGEAAQSVFDGDYDEIEIRQEWQEENTLHEWDEGEFQLEPPLDTEEGRAAADEWDER (SEQ ID NO: 5).

[0037] The amino acid sequence of the 6X His is as follows: HHHHHH.

[0038] The amino acid sequence of HRV3C is as follows: LEVLFQGP.

[0039] 2. Expression of fusion protein Cullin1-Rbx1

[0040] 2.1 The constructed recombinant vector MsyB-Cullin1-RBX1-His was transformed into BL21 (DE3) Escherichia coli by heat shock method and cultured on LB medium plates containing 50 μg / ml kanamycin overnight; then, a single colony was selected for sequencing identification;

[0041] 2.2 Take a positive single colony containing the recombinant plasmid and inoculate it into LB liquid medium containing a final concentration of 50 μg / ml kanamycin. Incubate it at 37°C and 200 rpm for 10 to 12 hours. Inoculate the cultured bacterial liquid into fresh LB liquid medium containing 50 μg / ml kanamycin at a volume ratio of 1:50. Incubate it at 37°C and 200 rpm until the bacterial liquid concentration OD600 is 0.8 to 1.2. Add IPTG (isopropyl-β-D-thiogalactoside) inducer to a working concentration of 0.2 mM. Continue to culture at 16°C and 180 rpm for 16 hours, centrifuge for 20 minutes, and collect the bacteria.

[0042] 2.3 Resuspend the cells: resuspend the cells collected by centrifugation in 50mM HEPES 150mM NaCl buffer (pH=7.5), add PMSF (phenylmethylsulfonyl fluoride) to a working concentration of 1mM, mix well, and then ultrasonically disrupt on ice. The ultrasonic parameters are: power 100W, ultrasonic 3s, interval 6s, a total of 40min; then centrifuge at 4℃, 12000rpm for 30min to obtain the supernatant containing protein;

[0043] 3. Purification of fusion protein Cullin1-Rbx1:

[0044] The supernatant was incubated with the nickel column and then washed and eluted with 20 mM and 300 mM imidazole. The eluate was collected and the protein molecular weight was verified by SDS-PAGE gel ( Figure 2 The protein was cleaved with PreScission protease to remove the His tag. The molecular weight of the Cullin1-Rbx1 protein after the His tag was removed is shown in ( Figure 2 As shown in (B), the eluate was dialyzed overnight in a dialysis buffer (the dialysis buffer contained 50 mM MES (2-(N-morpholino)ethanesulfonic acid) and 50 mM NaCl, pH 6.5); the next day, the sample was filtered through a 0.22 μm filter membrane and loaded onto a Source S 10 / 300GL column pre-equilibrated with a buffer (50 mM MES, pH 6.5), and eluted and separated with a linear gradient (0-50%) to obtain a pure Cullin1-Rbx1 fusion protein;

[0045] The protein concentration was determined by UV absorption and analyzed by SDS-PAGE electrophoresis; the electrophoresis results were shown in Figure 2 As shown in C. Using this method, 2 mg of protein can be purified per liter of bacteria, and the purity of the obtained Cullin1-Rbx1 fusion protein is as high as 99%. The isolated and purified protein was quickly frozen in liquid nitrogen and stored at -80°C for further use.

[0046] 4. Verify whether the fusion protein Cullin1-Rbx1 is active in an in vitro ubiquitination modification system:

[0047] A 50 μL reaction system was set up by adding MgCl to 50 mM HEPES 150 mM NaCl buffer (pH 7.5). 2 , ATP, UbC12, NAE1-UBA3, Nedd8 and Cullin1-Rbx1 fusion protein to obtain a reaction system, the content of each substance in the system is: 10mM MgCl 2 , 10mM ATP, 5μM UbC12, 1μM NAE1-UBA3, 50μM Nedd8 and 10μM Cullin1-Rbx1; the reaction system was incubated at 37℃ for 4 hours. After the incubation, samples were taken every 1 hour for SDS PAGE electrophoresis analysis ( Figure 3 In B), the electrophoresis results of Cullin1-Rbx1 alone were used for comparison. Figure 3 As shown in B.

[0048] from Figure 3 As shown in the results of Figure B, Nedd8 was successfully modified on Cullin1-Rbx1 in the in vitro ubiquitination modification system ( Figure 3 Middle A), demonstrating that the fusion protein Cullin1-Rbx1 is active.

[0049] The steps for preparing UbC12 (Uni Prot Entry No.: P61081) described in this example are as follows:

[0050] (1) Nanjing GenScript Biotechnology Co., Ltd. used the pGEX-6P-1 vector (which carries a GST tag) to construct an expression vector for UbC12. Specifically, the UbC12 gene fragment was used as a template to clone the UbC12 gene into the pGEX-6P-1 vector. The corresponding promoter was the Tac promoter and the restriction site was the Prescission Protease. The resulting recombinant vector was recorded as the pGEX-6P-1-UbC12 vector.

[0051] The amino acid sequence of the UbC12 gene is as follows:

[0052] MIKLFSLKQQKKEEESAGGTKGSSKKASAAQLRIQKDINELNLPKTCDISFSDPDDLLNFKLVICPDEGFYKSGKFVFSFKVGQGYPHDPPKVKCETMVYHPNIDLEGNVCLNILREDWKPVLTINSIIYGLQYLFLEPNPEDPLNKEAAEVLQNNRRLFEQNVQRSMRGGYIGSTYFERCLK (SEQ ID NO:6).

[0053] The DNA sequence of the UbC12 gene is shown below:

[0054] atgatcaagctgttctcgct gaagcagcag aagaaggagg aggagtcggc gggcggcaccaagggcagca gcaagaaggc gtcggcggcg cagctgcgga tccagaagga cataaacgag ctgaacctgcccaagacgtg tgatatcagc ttctcagatc cagacgacct cctcaacttc aagctggtca tctgtcctgatgagggcttc tacaagagtg ggaagtttgt gttcagtttt aaggtgggcc agggttaccc gcatgatccccccaaggtga agtgtgagac aatggtctat caccccaaca ttgacctcga gggcaacgtc tgcctcaacatcctcagaga ggactggaag ccagtcctta cgataaactc cataatttat ggcctgcagt atctcttcttggagcccaac cccgaggacccactgaacaa ggaggccgca gaggtcctgc agaacaaccg gcggctgtttgagcagaacg tgcagcgctc catgcggggt ggctacatcg gctccaccta ctttgagcgc tgcctgaaatag (SEQ ID NO:7).

[0055] (2) The pGEX-6P-1-UbC12 recombinant plasmid was transferred into E. coli BL21 (DE3) by heat shock method. The colonies grown after overnight culture were regarded as positive colonies. The positive single colony containing the recombinant plasmid was transferred to LB medium containing 100 µg / mL ampicillin and cultured at 37°C until OD600 reached 0.8. Then, 0.4 mM IPTG (final concentration) was added to induce expression at 16°C overnight. The cells were collected by centrifugation and sonicated in lysis buffer (50 mM HEPES, 150 mM NaCl, pH 8.0) (ultrasonication for 40 min, power 100 W). After centrifugation at 12,000 rpm for 30 min, the supernatant was loaded onto the GST column, the column was washed with wash buffer (50 mM HEPES, 150 mM NaCl, pH 8.0), and then eluted with elution buffer (50 mM HEPES, 150 mM NaCl, 25 mM GSH, pH 8.0) to elute the protein; then, the GST tag was removed using PreScission protease and the protein was concentrated to 10 mg / mL. 1 mL of the sample was loaded onto a Superdex™ 75 10 / 300GL column pre-equilibrated with A buffer (50 mM HEPES, 150 mM NaCl, pH 8.0) and separated at a flow rate of 0.4 mL / min for 63 minutes. The isolated and purified UbC12 protein was quickly frozen in liquid nitrogen and stored at -80°C for further use;

[0056] The preparation method of the NAE1-UBA3 (NAE1 Uni Prot Entry No.: Q13564 UBA3 Uni Prot Entry No.: Q8TBC4) protein described in this example is:

[0057] (1) Nanjing GenScript Biotechnology Co., Ltd. used the pGEX-6P-1 vector to construct an NAE1-UBA3 expression vector. The NAE1-UBA3 gene fragment was used as a template to clone the NAE1-UBA3 gene into the pGEX-6P-1 vector. The corresponding promoter was the Tac promoter, and the restriction site was the Prescission Protease. The resulting recombinant vector was named pGEX-6P-1-NAE1-UBA3.

[0058] The NAE1-UBA3 amino acid sequence is: MAQLGKLLKEQKYDRQLRLWGDHGQEALESAHVCLINATATGTEILKNLVLPGIGSFTIIDGNQVSGEDAGNNFFLQRSSIGKNRAEAAAMEFLQELNSDVSGSFVEESPENLLDNPSFFCRFTVVVATQLPESTSLRLADVLWNSQIPLLICRTYGLVGYMRIIIKEHPVIESHPDNALEDLRLDKPFPELREHFQSYDLDHMEKKDHSHTPWIVIIAKYLAQWYSETNGRIPKTYKEKEDFRDLIRQGILKNENGA PEDEENFEEAIKNVNTALNTTQIPSSIEDIFNDDRCINITKQTPSFWILARALKEFVAKEGQGNLPVRGTIPDMIADSGKYIKLQNVYREKAKKDAAAVGNHVAKLLQSIGQAPESISEKELKLLCSNSAFLRVVRCR SLAEEYGLDTINKDEIISSMDNPDNEIVLYLMLRAVDRFHKQQGRYPGVSNYQVEEDIGKLKSCLTGFLQEYGLSVMVKDDYVHEFCRYGAAEPHTIAAFLGGAAQEVIKIITKQFVIFNNTYIYSGMSQTSATFQL-MADGEEPEKKRRRIEELLAEKMAVDGGCGDTGDWEGRWNHVKKFLERSGPFTHPDFEPSTESLQFLLDTCKVLVIGAGGLGCELLKNLALSGFRQIHVIDMDTIDVSNLNRQFLFRPKDIGRPKAEVAAEFLNDRVPNCNVVPHFNKIQDFNDTFYRQFHIIVCGLDSIIARRWINGMLISLLNYEDGVLDPSSIVPLIDGGTEGFKGNARVILPGMTACIECTLELYPPQVNFPMCTIASMPRLPEHCIEYVRMLQWPKEQPFGEGVPLDGDDPEHIQWIFQKSLERASQYNIRGVTYRLTQGVVKRIIPAVASTNAVIAAVCATEVFKIATSAYIPLNNYLVFNDVDGLYTYTFEAERKENCPACSQLPQNIQFSPSAKLQEVLDYLTNSASLQMKSPAITATLEGKNRTLYLQSVTSIEERTRPNLSKTLKELGLVDGQELAVADVTTPQTVLFKLHFTS (SEQ ID NO:8).

[0059] The DNA sequence of the NAE1-UBA3 gene:

[0060] atggatg ctcagcaac aaaaacaat gaagccaggt tgtggggtga tcatgggcaagaggctttag aatctgctca tgttgccta ataatgca cagccacagg aactgaatt cttaaaaacttgtactacc aggtattggt tcgttacaa aacacaat aacaggaggtgg atgctggaaacaatttctcctc cttcaaagaa gcagtatcgg caaaccga gctgaagctg ccatggaatt cttacaagaatttaagcg atgtctctg aagtttgtg gagagagtc cagaaacct tctagacaat gatcctcattctctgcag gcagtcactgt tcactacgct tagcagatgtcctctggaat tcccagattc ctctttgat ctgtaggaca tatggactag ttggttatat gaggatcattaataaagaac atccagtaat agaatctcat ccagataatg cattagaga tctacgacta gataagccattctctgaact gagattcaggataccatttt acagtcatactccatggatt gtgatcatag ctaaatatttagcacagtgg tagtgaaa aaatggacg atacctaaacgtataaag aaaaagacttcagagat tgattagac aaggaattct aaaaaatgaa aatggggctccagagatgagagaattt gagaataacaagcagcat ctcagatcccaagcagtatt gaagatatat ttaatgatga tcgctgcata atatcacca aacagactcc atcattttggatttagctc gtgccttaaaggaatttgtg gccaaagagg gtcaaggaaa tttacctgtt cgaggcacaattcctgatat gattgcagat tcaggcaaat atataaaact gcaaaacgtt taccgtgaaa aagcaaagaaagatgctgcc gctgtgggta atcatgttgc caaattgctg cagtccacc agagcagcagtcagccattcag aattaaaatt actctgcagc aattctgcat ttcttcgagt ggtaagatgt cgatccttagctgaagaata tggtttggat acaattaaca aggatgaaat tatttctagc atggacaatc cagataatgaaatagtgttg tacttaatgt tacgggctgt tgatagattt cataaacaac agggtagata agaccagagattagtactactatc aagttgaagt cttgtctcac tggcttcctt caggaatatggtttatctgt aatggtgaaa gatgattatg tccacgaatt ttgccgatat ggagctgctg agccacataccattgctgca ttcttggggg gagctgctgc tcaagaggtc atcaaaataa tcaccaaaattt atttgtagtattagcattagcatta tcacaaactt cagcaacttt ccagttgtag -atggcggatggcgaggagcc aatggctgtt gatggtggggtggggacac tggagactgg gaaggtcgct ggaaccatgtaaagaagttc ctcgagcgat ctggaccctt cacacaccct gatttcgaac cgagcagtgactt agccttgaggtgattgaggtat gacactatag atgtttccaa tctaaatagg cagtttttatttaggcctaa agatattgga agacctaaggctgaagttgc tgcagaattt ctaaatgaca gagttcctaattgcaatgta gttccacatt tcaacagat tcaagatttt aacgactt tctatcgaca atttcatattattgtg gactggactc tatcatccgcc agagatgga taaatggcat gctgaatgttatgaccattacttct ttgtcccttt gatagatggg gggacagaag gttttaaggaaatgcccgg gtgattctgc ctggaatgac tgctgtatc gatgcacgc tggaacttta tccaccaggttaatttc ccatgtgtgcac cattgcaggagtact atgcccaggc taccagattagg tactgtagtag ctgtagc cagcctttg gagaaggggt tccattagat ggagatgatcctgaacatat acaatggatt ttccaaaat ccctagagag agcatcacaa tataatatta ggggtgttactataggctc actcagggg tagtaaag aatcattcct gcagctagct ccacaaatgaggggtgcttcattca ttttaaaata gccacaagtgcatacattcc cttgaataat tacttgtgtttaatgatgt agatgggctg tatacataca cattgaagc agaagaag gaaactgcc cagctgtagccagctcct caaatattc agttctctcc atcagctaatt tggataccatta tgtacatta tgcggaggg atctccagcc atcacagcca ccctagaggg aaaaaataga acacttacagtcggt aaccttatt gagaacgaa CAggccaaa tctcccaaaacattgaaag aattggggcttgttgatgga caagaactgg cggttgctga tgtcaccacc ccacagactg tactattcaa acttcattttacttcttaa (SEQ ID NO: 9).

[0061] (2) The pGEX-6P-1-NAE1-UBA3 plasmid was expressed in E. coli BL21 (DE3) using the heat shock method, and the cells were then cultured in LB medium containing 100 µg / mL ampicillin at 16°C until the OD600 reached 0.8, and then 0.4 mM IPTG (final concentration) was added to induce expression overnight at 16°C; the cells were then collected by centrifugation and lysed by sonication in lysis buffer (50 mM HEPES, 150 mM NaCl, pH 8.0); after centrifugation at 12,000 rpm for 30 min, the supernatant was loaded onto a GST column (Smart Lifesciences), the column was washed with wash buffer (50 mM HEPES, 150 mM NaCl, pH 8.0), and then eluted with elution buffer (50 mM HEPES, 150 mM NaCl, 25 mM GSH, pH 8.0). 8.0); then, the GST tag was removed using PreScission protease, filtered through a 0.22 µm filter membrane, loaded onto a Source Q 10 / 300GL column pre-equilibrated with A buffer (50 mM HEPES, pH 8.2), and eluted with a linear gradient from 0% to 50% B buffer (50 mM HEPES, 1 M NaCl, pH 8.2) at a flow rate of 1 mL / min for 30 min and concentrated to 1-2 mg / mL; the protein was quickly frozen in liquid nitrogen after SDS-PAGE electrophoresis analysis and stored at -80°C for further use.

[0062] The preparation method of Nedd8 (Uni Prot Entry No.: Q15843) protein described in this example is:

[0063] (1) The pET29a vector (synthesized by Genscript) was used by Nanjing GenScript Biotechnology Co., Ltd. to construct a Nedd8 expression vector. Specifically, the Nedd8 gene fragment was used as a template and the Nedd8 gene was cloned into the pET29a vector. The corresponding promoter was the T7 promoter and the restriction site was the Prescission Protease. The resulting recombinant vector was recorded as pET29a-Nedd8.

[0064] The amino acid sequence of the Nedd8 gene:

[0065] MLIKVKTLTGKEIEIDIEPTDKVERIKERVEEKEGIPPQQQRLIYSGKQMNDEKTAADYKILGGSVLHLVLALRGGGGLRQ (SEQ ID NO: 10).

[0066] The DNA sequence of the Nedd8 gene:

[0067] atgctgattaaggttaaaaccctgaccggcaaagagatcgaaattgacatcgagccgaccgataaggtggagcgtatcaaagaacgtgttgaggaaaaggaaggtattccgccgcag caacagcgtctgatctacagcggcaagcagatgaacgacgaaaaaaccgcggcggattataagattctgggtggcagcgtgctgcacctggttctggcgctgcgtggtggttaa (seq ID NO:11).

[0068] (2) The pET29a-Nedd8 plasmid was transformed into Escherichia coli BL21 (DE3) cells by heat shock method for expression, and then the cells were grown in LB medium containing 50 μg / mL kanamycin to OD600 of 0.6-0.8 and induced with a final concentration of 1 mM IPTG at 37°C for 6 hours; then, after centrifugation at 4000 rpm for 30 minutes at 4°C, the cells were resuspended in 50 mM HEPES, 150 mM NaCl buffer (pH 7.5), lysed by ultrasonication in an ice bath for 60 minutes, and centrifuged at 12000 rpm for 30 minutes; the precipitate after centrifugation was resuspended in 8 M urea aqueous solution (pH 8.0) and ultrasonicated for 30 minutes, and then centrifuged again at 12000 rpm for 30 minutes, and the supernatant was separated by C4 high-performance liquid chromatography column; the supernatant was dialyzed in dialysis buffer (50 mM HEPES, 150 mM NaCl, pH 7.5 ) and dialyzed against Superdex pre-equilibrated with a buffer containing 50 mM HEPES, 150 mM NaCl, pH 7.5. TM The purified Nedd8 protein was stored in liquid nitrogen at -80 °C and quickly frozen for further use.

[0069] 5. Verify whether the fusion protein Cullin1-Rbx1 can self-assemble into an E3 enzyme complex in vitro:

[0070] a. Set up a 50 μL reaction system, which is a reaction system obtained by adding the adaptor protein Skp1-Skp2, the receptor protein Cks1 and the fusion protein Cullin1-Rbx1 to 50 mM HEPES 150 mM NaCl buffer (pH 7.5), and the content of each substance in the system is: 1 μM Skp1-Skp2, 1 μM Cks1 and 1 μM Cullin1-Rbx1;

[0071] b. Incubate the reaction system on ice for 1 hour. After the reaction is complete, perform SDS PAGE electrophoresis analysis. The results are as follows: Figure 4 shown.

[0072] c.From Figure 4 It can be seen that it self-assembles into a Cullin1-Rbx1 E3 ligase complex in vitro.

[0073] The specific steps of the preparation method of Skp1-Skp2 (Skp1 Uni Prot Entry No.: P63208 Skp2 Uni Prot Entry No.: P63208) described in this embodiment are as follows:

[0074] (1) Nanjing GenScript Biotechnology Co., Ltd. used the pGEX-6P-1 vector (which comes with a GST tag) to construct an expression vector for Skp1-Skp2. Specifically, the Skp1-Skp2 gene fragment was used as a template to clone the Skp1-Skp2 gene into the pGEX-6P-1 vector. The corresponding promoter was the Tac promoter and the restriction site was the Prescission Protease. The resulting recombinant vector was recorded as pGEX-6P-1-Skp1-Skp2.

[0075] The amino acid sequence of the Skp1-Skp2 gene is as follows:

[0076] MPSIKLQSSDGEIFEVDVEIAKQSVTIKTMLEDLGMDDEGDDDPVPLPNVNAAILKKVIQWCTHHKDDPPPPEDDENKEKRTDDIPVWDQEFLKVDQGTLFELILAANYLDIKGLLDVTCKTVANMIKGKTPEEIRKTFNIKNDFTEEEEAQVRKENQWCEEK-MHRKHLQEIPDLSSNVATSFTWGWDSSKTSELLSGMGVSALEKEEPDSENIPQELLSNLGHPESPPRKRLKSKGSDKDFVIVRRPKLNRENFPGVSWDSLPDELLLGIFSCLCLPELLKVSGVCKRWYRLASDESLWQTLDLTGKNLHPDVTGRLLSQGVIAFRCPRSFMDQPLAEHFSPFRVQHMDLSNSVIEVSTLHGILSQCSKLQNLSLEGLRLSDPIVNTLAKNSNLVRLNLSGCSGFSEFALQTLLSSCSRLDELNLSWCFDFTEKHVQVAVAHVSETITQLNLSGYRKNLQKSDLSTLVRRCPNLVHLDLSDSVMLKNDCFQEFFQLNYLQHLSLSRCYDIIPETLLELGEIPTLKTLQVFGIVPDGTLQLLKEALPHLQINCSHFTTIARPTIGNKKNQEIWGIKCRLTLQKPSCL (SEQ ID NO:12).

[0077] The DNA sequence of the Skp1-Skp2 gene is as follows:

[0078] atgc cttcaattaa gttgcaattaa tctgatggag agatatttga agttgatgtggaaattgcca aacaatctgt gactattaag accatgttgg aagatttggg aatggatgat gaaggagatgatgacccagt tcctctacca aatgtgaatg cagcaatatt aaaaaaggtc attcagtcaggcacccactccatcc ctgaagatga tgagaacaaa gaaaagcgaa cagatgatat ccctgtttgggaccaagaat tcctgaaagt tgaccaagga acactttttg aactcattct ggctgcaaac tacttagacatcaaaggttt gcttgatgtt acatgcaaga ctgttgccaa gggaa tatgaccactcgcagatc ttcaatatca aaaatgactt tactgaagag gaggaagccc aggtaggtag cacacagttttgtctttga -atg gaccaaccat tggctgaaca tttcagtact ctcgcaaaaactcaaatttagtgcgactt aacctttctg ggtgttctgg attctctgaa tttgcccttggactgactggctcgc atgagctgaa cctctcctgg tgttttgatt tcactgaaaagcatgtacag gtggctgttg cgcatgtgtc agagaccatc acccagctga atcttagcgg ctacagaaagaatctccaga aatcagatct ctctacttta gttagaagat gccccaatct tgtccatcta gacttagactcat gcctgattgctcat aggaattttt ccagctcaac tacctccaac acctatcactcagtcggtgc tatgatataa tacctgaaac tttacttgaa cttggagaaattcccacact aaaaacactacaagtttttg gaatcgtgcc agatggtacc cttcaactgt taaaggaagc ccttcctcat ctacagattaattgctccca tttcaccacc attgccaggc caactattgg caacaaaaag aaccaggaga tatggggcatcaaatgccga ctgacactgc aaaagcccag ttgtctatga (SEQ ID NO: 13).

[0079] (2) The pGEX-6P-1-Skp1-Skp2 plasmid was expressed in E. coli BL21 (DE3), and then a single colony containing the recombinant positive plasmid was taken and cultured in LB medium containing 100 µg / mL ampicillin at 37°C until the OD600 reached 0.8, and then 0.4 mM IPTG (final concentration) was added to induce expression overnight at 16°C; the cells were then collected by centrifugation and lysed by ultrasonication in lysis buffer (50 mM HEPES, 150 mM NaCl, pH 8.0). After centrifugation at 12,000 rpm for 30 minutes, the supernatant was loaded onto a GST column; the column was washed with a wash buffer (50 mM HEPES, 150 mM NaCl, pH 8.0), and then eluted with an elution buffer (50 mM HEPES, 150 mM NaCl, 25 mM GSH, pH 8.0) to elute the protein; then, the GST tag was removed using PreScission protease, and the protein was concentrated to 10 mg / mL; 1 mL of the sample was loaded onto a Superdex™ 200 10 / 300GL column pre-equilibrated with buffer (50 mM HEPES, 150 mM NaCl, pH 8.0). The separation was performed at a flow rate of 0.4 mL / min for 63 minutes. The separated and purified protein was the Skp1-Skp2 protein, which was quickly frozen in liquid nitrogen and stored at -80°C for further use.

[0080] The preparation method of the Cks1 (Uni Prot Entry No.: P61024) protein described in this example is:

[0081] (1) Nanjing GenScript Biotechnology Co., Ltd. constructed a Cks1 expression vector using the pGEX-6P-1 vector (which has a GST tag). Specifically, the Cks1 gene fragment was used as a template to clone the Cks1 gene into the pGEX-6P-1 vector. The corresponding promoter was the Tac promoter and the restriction site was the Prescission Protease. The resulting recombinant plasmid was recorded as pGEX-6P-1-Cks1.

[0082] The amino acid sequence of the Cks1 gene is:

[0083] MSHKQIYYSDKYDDEEFEYRHVMLPKDIAKLVPKTHLMSESEWRNLGVQQSQGWVHYMIHEPEPHILLFRRPLPKKPKK (SEQ ID NO: 14).

[0084] The DNA sequence of the Cks1 gene is:

[0085] atgtc gcacaaacaa atttactatt cggacaaata cgacgacgag gagtttgagtatcgacatgt catgctgccc aaggacatag ccaagctggt ccctaaaacc catctgatgt ctgaatctgaatggaggaat cttggcgttc agcagagtca gggatgggtc cattatatga tccatgaacc agaacctcacatcttgctgt tccggcgccc actacccaag aaaccaaaga aatga (SEQ ID NO: 15).

[0086] (2) The pGEX-6P-1-Cks1 plasmid was expressed in E. coli BL21 (DE3) by heat shock method, and then a single positive colony containing the recombinant plasmid was taken and cultured in LB medium containing 100 µg / mL ampicillin at 37°C until OD600 reached 0.8, and then 0.4 mM IPTG (final concentration) was added to induce expression overnight at 16°C; the cells were collected by centrifugation and lysed by ultrasonication in lysis buffer (50 mM HEPES, 150 mM NaCl, pH 8.0), and after centrifugation at 12000 rpm for 30 minutes, the supernatant was loaded onto the GST column; the column was washed with washing buffer (50 mM HEPES, 150 mM NaCl, pH 8.0), and then eluted with elution buffer (50 mM HEPES, 150 mM NaCl, 25 mM GSH, pH 8.0) to elute the protein; then, the GST tag was removed using PreScission protease and the protein was concentrated to 10 mg / mL; 1 mL of the sample was loaded onto a Superdex™ 75 10 / 300GL chromatography column that had been pre-equilibrated with buffer (50 mM HEPES, 150 mM NaCl, pH 8.0), and separation was performed at a flow rate of 0.4 mL / min for 63 minutes. The separated and purified protein was the Cks1 protein, which was quickly frozen in liquid nitrogen and stored at -80°C for further use.

Claims

1. A method for efficiently obtaining Cullin1-Rbx1 E3 ligase complex by prokaryotic expression, characterized in that: The specific steps are as follows: (1) Obtaining Cullin1-Rbx1 fusion protein by prokaryotic expression: 1) Construction of recombinant vector MsyB-Cullin1-Rbx1-His: The human Cullin1 gene was codon optimized, and the MsyB solubilization tag and HRV 3C restriction site were added to its N-terminus, and then the optimized Cullin1 was cloned behind the first ribosome binding site of the pET29a vector; then, the human Rbx1 gene was codon optimized, and the 6X His tag and HRV 3C restriction site were added to its N-terminus, and then it was cloned behind the second ribosome binding site of the pET29a vector to obtain the recombinant vector MsyB-Cullin1-Rbx1-His; the amino acid sequence of the Cullin1 gene is SEQ ID NO: 2; the amino acid sequence encoded by the RBX1 gene is SEQ ID NO: 4; the amino acid sequence of the MsyB is SEQ ID NO: 5; the amino acid sequence of the 6X His is HHHHHH; the amino acid sequence of the HRV3C is as follows: LEVLFQGP; 2) Expression of fusion protein Cullin1-Rbx1: The recombinant vector MsyB-Cullin1-Rbx1-His was transformed into Escherichia coli BL21 (DE3) by heat shock method and cultured overnight on LB plates containing kanamycin; then a single positive colony containing the recombinant plasmid was taken and inoculated into LB liquid medium containing kanamycin, and cultured with shaking for 10 to 12 hours. The cultured bacterial liquid was inoculated into fresh LB liquid medium containing kanamycin, and cultured with shaking until the bacterial liquid concentration OD600 was 0.8 to 1.2, and then the inducer isopropyl-β-D-thiogalactoside was added. After continuing to culture for 10-26 hours, centrifugation was performed to collect and resuspend the bacterial cells; The collected bacteria were resuspended in 50mM HEPES 150mM NaCl buffer, phenylmethylsulfonyl fluoride was added, mixed, and ultrasonically disrupted on ice; then centrifuged to obtain the supernatant containing protein; 3) Purification of fusion protein Cullin1-Rbx1: Purification was performed using a nickel column. The supernatant was incubated with the nickel column and then washed and eluted with imidazole. The eluate was collected and the protein was cleaved with PreScission protease to remove the His tag. The eluate was dialyzed in dialysis buffer overnight. The next day, the sample was filtered with a filter membrane and loaded onto a Source S 10 / 300GL column for linear gradient elution and separation to obtain a pure Cullin1-Rbx1 fusion protein. (2) Preparation of Cullin1-Rbx1 E3 ligase: The protein components, adaptor proteins Skp1-Skp2, Cks1 and Cullin1-RBX1, were reacted in a reaction system under the following reaction conditions: incubation on ice for one hour to obtain a Cullin1-Rbx1 E3 ligase complex.

2. The method for efficiently obtaining Cullin1-Rbx1 E3 ligase complex by prokaryotic expression according to claim 1, characterized in that: The reaction system also contains 50 mM HEPES 150 mM NaCl buffer, and the contents of various substances in the system are: 1 μM Skp1-Skp2, 1 μM Cks1 and 1 μM Cullin1-Rbx1.

3. The method for efficiently obtaining Cullin1-Rbx1 E3 ligase complex by prokaryotic expression according to claim 1, characterized in that: The final concentration of kanamycin in LB plates and LB liquid medium was 50 μg / ml.

4. The method for efficiently obtaining Cullin1-Rbx1 E3 ligase complex by prokaryotic expression according to claim 1, characterized in that: Step 2) The shaking culture conditions are: 37°C, 200 rpm.

5. The method for efficiently obtaining Cullin1-Rbx1 E3 ligase complex by prokaryotic expression according to claim 1, characterized in that: The parameters of the ultrasonic disruption are: power 100 W, ultrasonication 3 s, interval 6 s, total 40 min.

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