An eif3 octamer mutant and a method for preparing the same

Abstract

The application belongs to the field of genetic engineering, and provides an engineered eIF3 octamer core complex.The preparation method comprises the following steps: obtaining polypeptides with mutated eIF3 core subunits, including but not limited to eIF3a, eIF3c, eIF3e, eIF3k, eIF3l, eIF3m, eIF3f and eIF3h; after polypeptide folding to form a subunit, the subunit is assembled into an eIF3 core octamer; and the mutation is selected from the eIF3e:R376C, eIF3l:S462C, eIF3e:Y401C, eIF3l:Y539C, eIF3f:S346C and eIF3l:F550C sites.In the application, a disulfide bond is introduced between the eIF3k, eIF3e and eIF3f subunits, the interaction between the eIF3k / l subunit and the eIF3 core complex is enhanced, and the stability of the eIF3 core octamer is improved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to modified protein polymers, specifically to an engineered eIF3 octomer core complex. This invention also relates to the preparation method and application of this eIF3 octomer core complex. Background Technology

[0002] Translation initiation in eukaryotes is quite complex, consistent with the intricate gene regulation mechanisms of eukaryotes. eIF3 (eukaryotic initiation factor 3) is the largest translation initiation factor in eukaryotes, playing multiple roles in the assembly of the 43S preinitiation complex (PIC) and participating in almost the entire translation initiation process. It not only participates in the recognition of the start codon but also acts as a "scaffold" protein through interactions with various factors, assisting in the loading of the 40S small subunit onto mRNA, etc.

[0003] In mammals, eIF3 is composed of 13 subunits (eIF3a - eIF3m). According to published eIF3 complex structure data (Structure of a human 48S translational initiation complex. Science, 2020, 369(6508): 1220-1227), eight subunits of eIF3 (a, c, e, k, l, m, f, and h) constitute the eIF3 core octamer (Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression. ACS Synthetic Biology, 2023, 12(1): 136-143), while the other five subunits are relatively flexibly connected to the core subunit. The eIF3 core octamer shares a similar octamer structure with the proteasome lid and COP9 signalosome, sharing a common domain called the PCI (proteasome-COP9 signalosome-eIF3, PCI) domain. Subunits containing the PCI domain (a, c, e, l, k, and m) are arranged sequentially to form an arc-shaped structure. MPN subunits (f and h) bind to each other and attach to the rest of the octamer primarily through the association between eIF3f and eIF3m. In addition to the PCI arc, at least one α-helix of each subunit (except eIF3a and eIF3m) participates in the formation of a hepta-helical bundle, ultimately forming the classic five-lobed structure.

[0004] However, because the eIF3k / l subunit is relatively easy to detach from the eIF3 core complex, it affects the interaction between the eIF3 core complex and eIF4G and eIF4F to recruit mRNA, thus greatly impacting the in vitro translation efficiency of mRNA. Therefore, increasing the stability of the eIF3 core octamer complex through mutation has become an important means to improve the in vitro translation efficiency of mRNA. Summary of the Invention

[0005] The technical problem to be solved by this invention is to reinforce the eIF3 core octamer complex.

[0006] The second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned reinforced eIF3 core octamer complex.

[0007] Strengthening the spatial structure of protein subunits can be achieved through various methods, including the use of small molecule cross-linking agents, glycosylation modification, insertion of other amino acids, addition of natural substrates, inhibitors, cofactors or artificial ligands, salt bridges, optimization of hydrogen bond networks, and optimization of the protein's solvent environment. However, tests on the eIF3 octamer revealed that the most common small molecule cross-linking agents can affect the structure of the eIF3 octamer and the assembly of the transcription initiation complex, while optimization of the protein's solvent environment is not stable enough. This invention, after repeated testing, found that introducing three different sets of disulfide bonds at the α-helix and β-sheet ends of eIF3e and eIF3l can effectively enhance the binding strength between the two subunits of eIF3k / l (or collectively referred to as eIF3kl) and the hexamer of eIF3c / h / f / a / e / m (or collectively referred to as eIF3chfaem), with minimal impact on other subunits and their functions. Through repeated testing, the disulfide bond positions selected in this invention avoid the interaction regions between eIF3 and other translation initiation complexes. These positions are close together, allowing for efficient disulfide bond formation and reducing the impact of introducing disulfide bonds on the structure of eIF3 itself and the assembly of the translation initiation complex. This invention is based on the aforementioned research.

[0008] In a first aspect, the present invention provides an eIF3 octamer mutant. Disulfide bonds are introduced into each subunit of the eIF3 octamer through mutation.

[0009] For example, three pairs of disulfide bonds are introduced between the three subunits eIF3k, eIF3e, and eIF3f. Preferably, the eIF3 octamer mutant contains one or more of the following mutations: eIF3e:R376C, eIF3l:S462C; eIF3e:Y401C, eIF3l:Y539C; eIF3f:S346C, eIF3l:F550C.

[0010] This invention introduces three pairs of disulfide bonds between the three subunits eIF3k, eIF3e, and eIF3f to enhance the interaction between the eIF3kl subunit and the eIF3 core complex, thereby improving the stability of the eIF3 core octamer. Using a ribozyme-mediated polycistronic co-expression plasmid system (ribozyme-assisted polycistronic, pRAP), the eIF3chfaem hexamer and eIF3kl dimer were expressed in an E. coli expression system and successfully assembled into a complete eIF3 octamer complex. Experiments verified the disulfide bond formation and changes in thermal stability of the mutants using non-reducing SDS-PAGE and protein stability analysis. None of the three mutations significantly affected the complex structure or purification process, while simultaneously enhancing the stability of the eIF3 core octamer to some extent. The eIF3 core complex containing eIF3e:R376C and eIF3l:S462C exhibited a good state under cryo-electron microscopy.

[0011] Secondly, the present invention provides a method for preparing the above-mentioned eIF3 octamer mutant.

[0012] In short, the preparation method includes: obtaining protein components with mutated eIF3 core subunits, wherein the eIF3 core subunits include eIF3a, eIF3c, eIF3e, eIF3k, eIF3l, eIF3m, eIF3f, and eIF3h. The above protein subunits are assembled into an eIF3 core octamer. The mutations described in this invention are selected from one or more of the following groups: eIF3e: R376C, eIF3l: S462C; eIF3e: Y401C, eIF3l: Y539C; eIF3f: S346C, eIF3l: F550C sites.

[0013] Obtaining a polypeptide with a mutated eIF3 core subunit involves expressing the nucleic acid encoding the mutated eIF3 core subunit as a corresponding protein. The protein polypeptide can be artificially synthesized according to a predetermined amino acid sequence, or expressed as the corresponding amino acid after artificial synthesis of its encoding nucleic acid. Preferably, the nucleic acid encoding the mutated eIF3 core subunit is obtained by amplifying a plasmid containing the nucleic acid encoding the eIF3 core subunit using specially designed primers. In a preferred embodiment of the invention, six pairs of primers are designed, as shown in Table 1.

[0014] When designing primers, the mutation site is placed in the center of the complementary primer, with 5-10 bases extending on each side to ensure 15-20 base pairs between the two primers. Each primer is then extended 5-10 bases at its 5' end to ensure primer specificity. The entire plasmid is amplified using polymerase chain reaction (PCR). In a preferred embodiment of this invention, the PCR reaction system amplification procedures are shown in Tables 9 and 10, respectively.

[0015] Assembling the eIF3 core octamer involves mixing the protein components of the eIF3 core subunits in a specific ratio, incubating on ice, and then obtaining the eIF3 core octamer. The specific process includes: first obtaining an equimolar amount of eIF3chfaem hexamer, and then mixing it with eIF3kl dimer, or directly mixing individual core octamer subunits. During mixing, the molar numbers of eIF3c / h / f / a / e / m subunits are equal, the molar numbers of eIF3k / l subunits are equal, and the molar ratio of eIF3c to eIF3k subunits is 1:(1-3), preferably 1:(1.1-2), and more preferably 1:(1.2-1.5). After incubating the eIF3 core octamer at 0-5℃ for 30 minutes, centrifuge at no more than 12000 rpm for 5-10 minutes to obtain the supernatant. Using a protein purification system, inject the mixture sample through a loading loop and collect the elution fraction. Based on the SDS-PAGE gel running results and protein purification peak chromatogram of the elution fraction, select A... 280 With A 260 Samples with the correct molecular weight in the peak were concentrated to the ideal concentration by ultrafiltration and then centrifuged to obtain the supernatant of the eIF3 core octomer.

[0016] The preparation method of the present invention includes multi-step protein purification. Conventional protein purification methods in the art can be used, namely: prokaryotic overexpression of protein, high-pressure disruption, nickel affinity chromatography, gel filtration chromatography, etc.

[0017] The general procedure for high-pressure cell disruption is as follows: After resuspending the bacterial culture overexpressing the eIF3 core subunit with binding buffer, add the protease inhibitor Cocktail and PMSF solution, mix well, and then disrupt the bacterial cells using a high-pressure cell disruptor at 800 bar. Stop pressurizing when the color of the solution at the outlet changes from off-white to transparent, and collect the lysed bacterial culture. Centrifuge at a speed of not less than 12,000 rpm at 0-4℃ for not less than 40 minutes, and collect the lysed supernatant.

[0018] The general procedure for nickel affinity chromatography is as follows: Lysis buffer containing the eIF3 core subunit is loaded into a pre-packed column, and the flow-through is collected; the protein purification system is turned on, and the pre-packed column is connected to the system dropwise; the pre-packed column is washed with binding elution buffer until A... 280 Approaching baseline; elute with an imidazole concentration gradient using elution buffer, and wait for A 280 Once the rise begins, the eluent is collected.

[0019] The general procedure for gel filtration chromatography is as follows: The lysis buffer containing the eIF3 core subunit is concentrated to within 10 mL using an ultrafiltration tube, centrifuged at 12000 rpm and 4°C for 5 minutes to remove the precipitate, retaining the supernatant; a loop is loaded onto a protein purifier, the loop is washed with a threaded syringe, and the protein sample containing the eIF3 core subunit is loaded onto the loop; the protein sample is loaded onto a pre-packed column using the protein purifier, and the collection program is started. Finally, the desired fraction is selected based on the peak chromatogram and SDS-PAGE protein gel results, and then concentrated and stored.

[0020] In a preferred embodiment of the present invention, the preparation method includes: point mutation of a plasmid containing nucleic acid encoded by the eIF3 core subunit, followed by transformation into a recombinant plasmid to express the target protein (eIF3 core subunit); amplification, collection, and purification of the target protein; and assembly of the complex to form a stable, engineered eIF3 core complex. The engineered eIF3 core complex can then be cryopreserved or identified.

[0021] Thirdly, the present invention also provides applications of the above-described preparation method.

[0022] Using the above-described preparation method, this invention obtained an engineered eIF3 octamer core complex, improving the stability of the eIF3 octamer. Tests conducted in this invention show that all three mutant groups can form disulfide bonds, which not only enhances the interaction strength between eIF3kl and eIF3chfaem but also promotes the stability of eIF3 to a certain extent. Furthermore, the mutations did not affect the folding state of the eIF3 core complex.

[0023] Two-dimensional classification results showed that most of the mutant eIF3 core octamer contained the complete eIF3 "five-lobed" structure, and the mutant eIF3 core octamer was functionally active. The preparation method of this invention improves the accuracy of eIF3 core complex analysis in the initiation complex, contributing to a better understanding of the role of eIF3 in translation initiation.

[0024] The eIF3 octamer mutant of the present invention can be used to prepare an mRNA translation kit, wherein the mRNA translation kit contains the above-mentioned eIF3 octamer mutant.

[0025] In the description of this application, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Relational terms such as "first," "second," etc., are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

[0026] As used herein, the term “about” typically means + / - 5% of the value, more typically + / - 4% of the value, more typically + / - 3% of the value, more typically + / - 2% of the value, more typically + / - 1% of the value, and even more typically + / - 0.5% of the value.

[0027] Because the eIF3k / l subunit readily detaches from the eIF3 core complex, it affects the interaction between the eIF3 core complex and eIF4G and eIF4F to recruit mRNA, thus hindering the elucidation of related mechanisms. Although low-resolution structural models exist to infer the location of eIF4G on the eIF3 core complex, direct evidence for the eIF3-eIF4F-mRNA interaction structure remains lacking. This invention optimizes the structural stability of the human eIF3 octamer core complex through an engineering modification strategy. The stabilized complex will help improve the resolution of the eIF3 core complex, provide material for eIF3-eIF4G and eIF3-eIF4F-mRNA interactions, and contribute to elucidating the function of eIF3 in eukaryotic initiation. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, each drawing described below is for a part of the embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 This is a structural diagram of the eIF3 core octamer. Each subunit is labeled with a color, and mutation sites are represented by corresponding colored spheres at their respective locations in the diagram.

[0030] Figure 2This is the purification chromatogram and SDS-PAGE analysis of eIF3chfaem hexamer. The eIF3chfaem nickel affinity chromatography purification chromatogram shows the sample containing the eIF3e (R376C) mutation; the red line is A260nm, the blue line is A280nm, and the green line is the His B Buffer concentration. (B) and (C) SDS-PAGE corresponding to the elution peaks of the nickel affinity chromatography column. M, protein marker; T, bacterial cell sonication lysate; S, supernatant after centrifugation of the lysate; P, precipitate after centrifugation of the lysate; FL, flow-through buffer after passing the lysate through the nickel affinity chromatography column; 1A1-2B5 are the corresponding tube numbers for the nickel affinity chromatography eluent. (D) Gel filtration chromatography purification chromatogram shows the sample containing the eIF3e (R376C) mutation; the red line is A260nm, and the blue line is A280nm. (E) SDS-PAGE corresponding to the elution peaks of the gel filtration chromatography column. M, protein marker; Input, sample loaded for gel filtration chromatography; 1B3-2C3 are the collection tube numbers corresponding to the gel filtration chromatography eluent: 1B3-1C1 corresponds to the first peak in Figure D; 1C2-2A1 corresponds to the second peak in Figure D, marked with an asterisk; 2A4-2C3 corresponds to the third peak in Figure D; the molecular weight of each eIF3 subunit is marked on the right side of Figures B, C, and E.

[0031] Figure 3 This is a purification chromatogram and SDS-PAGE analysis of eIF3kl dimer. (A) EIF3kl nickel affinity chromatography purification chromatogram, the sample contains the eIF3l (S462C) mutation; the red line is A260nm, the blue line is A280nm, and the green line is the His B Buffer concentration. (B) SDS-PAGE corresponding to the elution peaks of the nickel affinity chromatography column. M, protein marker; T, bacterial cell sonication lysate; S, supernatant after centrifugation of the lysate; P, precipitate after centrifugation of the lysate; FL, flow-through buffer after passing the lysate through the nickel affinity chromatography column; 1A1-1B5 are the tube numbers corresponding to the collection tubes of the nickel affinity chromatography eluent. (C) Gel filtration chromatography purification chromatogram, the sample contains the eIF3l (S462C) mutation; the red line is A260nm, the blue line is A280nm, and the brown line is conductivity. (D) SDS-PAGE corresponding to the elution peaks of the gel filtration chromatography column. M, protein marker; 2C5-4C1 are the collection tube numbers corresponding to the gel filtration chromatography eluent: 2C5-3C1, corresponding to the second peak in Figure C; 4C1 corresponds to the third peak in Figure C; the molecular weight of eIF3kl is marked on the right side of Figures B and D.

[0032] Figure 4This is a purification spectrum of the eIF3 core octomer and its SDS-PAGE analysis. (A), (C), and (E) are purification spectra of the eIF3 core octomer, corresponding to (B), (D), and (F) respectively. The sample contains eIF3l and eIF3e mutations, with the mutation sites marked in the figure; the red line is A260nm, and the blue line is A280nm. (B), (D), and (F) are SDS-PAGE images corresponding to the elution peaks of the gel filtration chromatography column. M stands for protein marker; Input is the loaded sample; 1B1-1C3 are the corresponding tube numbers for nickel affinity chromatography eluents.

[0033] Figure 5 This is an SDS-PAGE analysis of the eIF3 core octamer. M represents the protein marker; lanes 1 and 2 contain the wild-type full-length eIF3 core octamer; lanes 3 and 4 contain the eIF3e:R376C and eIF3l:S462C mutations; lanes 5 and 6 contain the eIF3e:Y401C and eIF3l:Y539C mutations; lanes 7 and 8 contain the eIF3f:S346C and eIF3l:F550C mutations; lanes 1, 3, 5, and 7 used protein loading buffer with a reducing agent; lanes 2, 4, 6, and 8 used protein loading buffer without a reducing agent.

[0034] Figure 6 This is a thermal stability test of the eIF3 core octamer. Wide type: the natural eIF3 core octamer, truncated in the same form as the mutant; mutation 1: the eIF3 core octamer with the mutations eIF3e:R376C and eIF3l:S462C; mutation 2: the eIF3 core octamer with the mutations eIF3e:Y401C and eIF3l:Y539C; mutation 3: the eIF3 core octamer with the mutations eIF3f:S346C and eIF3l:F550C; A and B are respectively A... 330nm A 350nm Fluorescence intensity as a function of temperature; C represents A 350nm / A 330nm Fluorescence intensity ratio; D is A 350nm / A 330nm The fluorescence intensity ratio is obtained by fitting the first derivative of the Boltzmann equation, and the x-value corresponding to the highest point is T. m value.

[0035] Figure 7These are cryo-electron microscopy images of the eIF3 core octamer. (A) 300 kV electron microscopy image of the natural eIF3 core octamer; (B) 300 kV electron microscopy image of the eIF3 core octamer with the mutations eIF3e:R376C and eIF3l:S462C; (C) 200 kV electron microscopy image of the eIF3 core octamer with the mutations eIF3e:Y401C and eIF3l:Y539C; (D) 200 kV electron microscopy image of the eIF3 core octamer with the mutations eIF3f:S346C and eIF3l:F550C.

[0036] Figure 8 These are the two-dimensional classification results of the eIF3 core octamer by cryo-electron microscopy. (A) Natural eIF3 core octamer; (B) eIF3 core octamer with the mutations eIF3e:R376C and eIF3l:S462C.

[0037] Figure 9 The images show cryo-electron microscopy (cryo-EM) images, two-dimensional classification, data processing flowcharts, gold standard FSC curves, and angular distribution diagrams of the eIF3 core complex. (A)-(C) represent the cryo-EM images, two-dimensional classification, and data processing flowcharts of the eIF3 core complex, respectively.

[0038] Figure 10 This is the result of cryo-electron microscopy two-dimensional classification of eIF3-eIF4G. Detailed Implementation

[0039] 1. In this invention, the accession numbers of the eIF3 core octomer subunit in the UniProt (Universal Protein Resource) database are as follows: eIF3a is Q14152, eIF3c is Q99613, eIF3e is P60228, eIF3k is Q9UBQ5, eIF3l is Q9Y262, eIF3m is Q7L2H7, eIF3f is O00303, and eIF3h is O15372. The plasmids used in this invention containing the human translation initiation factor eIF3 subunits a (1-606), c (321-913), e, m, f (84-357), h (34-352), k, and l genes were all prepared according to the article "Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression." (ACS Synthetic Biology, 2023, 12(1): 136-143). Unlabeled sequences are full-length.

[0040] 2. Materials and Methods 2.1 Experimental Materials, Instruments, Reagents, and Consumables Unless otherwise specified, this invention uses experimental materials, instruments and reagents commonly used in the field.

[0041] 2.1.1 Experimental Materials The point mutations in the human translation initiation factor eIF3 and the primer sequences required for sequencing were provided by Beijing Qingke Biotechnology Co., Ltd. The specific sequences are shown in Table 1. Other experimental materials used in this invention are shown in Table 2: 2.1.2 Experimental Apparatus 2.1.3 Experimental Consumables 2.1.4 Experimental Reagents and Preparation Methods .

[0042] The commonly used reagent preparation schemes of this invention are as follows: The commonly used reagent preparation schemes of this invention are as follows: (1) Preparation of partial solution mother liquor The preparation schemes for some of the mother liquor solutions are shown in Table 6: ; (2) LB liquid culture medium (1 L) Take 25 g of LB premixed powder, add RO water to a final volume of 1 L, sterilize using the liquid sterilization program (121℃, 20 minutes), and use after cooling. (3) LB solid culture medium (200 mL) Take 5 g of LB premixed powder, 2.5 g of agar powder, and bring the volume of RO water to 200 mL. Sterilize using the liquid sterilization program (121℃, 20 minutes). When the temperature drops to 55℃, add the corresponding antibiotic stock solution according to the ratio, dispense into plates, and place in a 4℃ refrigerator after solidification. (4) 1 M HEPES-K, pH 7.4 (1 L) Accurately weigh 238.3 g of HEPES solid powder, dissolve it in 800 mL of ultrapure water, add about 25 g of solid KOH, adjust the pH to 7.4, and finally bring the volume to 1 L. Filter the solution through a 0.22 μm surface filter membrane and store it at low temperature (4℃). (5) 20×MOPS electrophoresis buffer Accurately weigh 208 g MOPS solid powder, 121.2 g Tris-base solid powder, 20 g SDS solid powder, and 6 g EDTA solid powder, completely dissolve them in 800 mL of ultrapure water, and finally bring the volume to 1 L. Filter the solution through a 0.22 μm surface filter membrane and store it at room temperature away from light. (6) 4×SDS-PAGE loading buffer (50 mL) For reduced 4×SDS-PAGE loading buffer, accurately weigh 1.514 g Tris-base, 20 mL glycerol, 3.5 mL 14.3 M β-Me, 0.2 g bromophenol blue, and 4 g SDS solid powder, dissolve them thoroughly in ultrapure water, and bring the volume to 50 mL. Aliquot and store at -20°C. For non-reduced 4×SDS-PAGE loading buffer, omit β-Me, bring the volume to 50 mL, aliquot, and store at -20°C.

[0043] 2.2 Experimental Methods 2.2.1 Plasmid point mutation experiment Because it was necessary to replace individual amino acids in the eIF3l, eIF3e, and eIF3f subunits, the codons of specific amino acid sites on the protein were changed to replace cysteine ​​codons. After repeated testing, six pairs of alteration sites were finally selected, as shown in Table 8. When designing primers, the mutation sites were placed in the center of the complementary primers, with 5-10 bases extended on each side to ensure 15-20 base pairs between the two primers. Each primer was then extended 5-10 bases at its 5' end to ensure primer specificity. The entire plasmid was amplified using polymerase chain reaction (PCR). The PCR reaction procedures are shown in Tables 9 and 10, respectively. After the reaction, the products were detected by 0.8% agarose gel electrophoresis, and 0.2 μL of DpnI enzyme was added at 37°C to digest the methylated original plasmid template. .

[0044] 2.2.2 Transformation of recombinant plasmids The digestion product was transformed into DH5α competent cells using a heat shock method. The specific steps were as follows: First, 100 μL of DH5α competent cells were removed from a -80°C cryogenic freezer and slowly thawed on ice. After complete thawing, 2 μL of the digestion product was added to the competent cells, gently tapped to mix, and then incubated on ice for 10 minutes. The mixture was then transferred to a 42°C metal bath for 90 seconds. Immediately afterward, the competent cells were returned to ice and incubated for 10 minutes, avoiding shaking. Next, 700 μL of antibiotic-free LB liquid medium was added, and the cells were cultured in a shaking incubator at 37°C and 220 rpm for 1 hour. Afterward, the cells were centrifuged, and the 700 μL supernatant was discarded. The cell pellet was resuspended in the remaining 100 μL of medium. Finally, 50 μL of the resuspended bacterial solution was evenly spread onto LB agar plates containing the appropriate antibiotic, and the plates were incubated overnight at 37°C.

[0045] 2.2.3 Identification and Extraction of Recombinant Plasmids Three clones were randomly selected from the plate and added to 5 mL of LB liquid medium containing the corresponding antibiotic. The plates were then incubated overnight at 37°C with a shaking incubator at 220 rpm. The next day, 1 mL of the bacterial culture was sent for analysis. Plasmids were extracted from the remaining bacterial cultures using the Novizan plasmid DNA miniprep kit. The procedure was as follows: The equilibrated centrifuge tube was centrifuged at 10,000 rpm for 1 minute, the medium was discarded, and the tube was inverted on absorbent paper for 5 minutes. Then, Buffer P1, Buffer P2, and Buffer P3 were added sequentially in a 5:5:7 ratio. Buffer P1 was used to resuspend the bacterial cells, lyse the bacterial culture, and neutralize Buffer P2, respectively. The tube was gently inverted to mix thoroughly. The tube was then centrifuged at 12,000 rpm for 10 minutes, and the supernatant was transferred to an adsorption column. The column was centrifuged at 12,000 rpm for 2 minutes, and the flow-through was discarded. Next, 700 μL of PW2 was added, and the column was washed twice by centrifugation at 12,000 rpm for 1 minute. The column was then centrifuged at 12,000 rpm for 2 minutes and dried. After standing at room temperature for 5 minutes to allow the alcohol to completely evaporate, add 50 μL of preheated ultrapure water (50℃) to the adsorption column. Once the DNA is completely dissolved, centrifuge at 12000 rpm for 2 minutes and transfer the plasmid solution to a new 1.5 mL EP centrifuge tube. Finally, determine the concentration using NanoDrop One and analyze the extracted recombinant plasmid using 0.8% agarose gel electrophoresis. Based on the results, store the plasmid with the correct point mutation at -20℃.

[0046] 2.2.4 High-level expression of the target protein Two target plasmids, one containing kanamycin resistance and the other ampicillin resistance, were transformed into HI-Control (DE3) pRARE2 competent cells. Cells expressing the two plasmids were selected using plates containing four antibiotics (kanamycin, ampicillin, gentamicin, and chloramphenicol; the latter two are required for competence. Single colonies were picked and transferred to 100 mL of medium containing the four antibiotics, and incubated overnight at 220 rpm and 37°C. Then, 10 mL of the bacterial culture was transferred to 1 L of LB broth containing the four antibiotics and incubated at 220 rpm and 37°C for approximately 3 hours, with OD measured every 30 minutes. 600 OD of bacterial culture 600 Once the culture reaches 0.6, stop the incubation and place the conical flask in a 4°C cool room for 30 minutes. After the bacterial culture has completely cooled, add 100 μL of 1M IPTG per liter of bacterial culture and incubate at 220 rpm and 18°C ​​for approximately 15 hours.

[0047] 2.2.5 Collection of Escherichia coli Pour the overnight bacterial culture into a 1 L ultracentrifuge bottle, balance the liquid, and place it in an Optima XPN-100 Ultracentrifuge floor-standing centrifuge. Centrifuge at 5000 rpm, 4°C for 15 minutes, discard the supernatant, transfer the wet culture to a 50 mL centrifuge tube, wash the cells with 1×PBS, balance the liquid, and place it in a Centrifuge 5810R centrifuge. Centrifuge at 4000 rpm, 4°C for 30 minutes, discard the supernatant, flash-freeze in liquid nitrogen, and store in an ultra-low temperature incubator at -80°C.

[0048] 2.2.6 Purification of the target protein The buffer solutions used in this step are shown in Table 11. All solutions were filtered using a 0.22 μm surface filter membrane. (1) High-pressure crushing Resuspend the bacterial cells in 5-10 times binding buffer, add a protease inhibitor cocktail and a final concentration of 1 mM PMSF solution at a ratio of 1:100, and mix well. Rinse the pre-cooled UH-06 autoclave with approximately 50 mL of 0.2 M NaOH, 1 L of ultrapure water, and 100 mL of binding buffer sequentially. Add the reselected bacterial culture to the feed cup, and after the air bubbles are expelled, pressurize to 800 bar. Stop pressurizing when the solution at the outlet changes from off-white to clear, and collect the lysis buffer. Transfer the lysis buffer to 40 mL high-speed centrifuge tubes, balance, and place in an Optima XPN-100 Ultracentrifuge floor-standing centrifuge. Centrifuge at 17000 rpm, 4°C for 60 minutes, collect the supernatant for subsequent protein purification, and reserve a small amount of lysis buffer for SDS-PAGE analysis. After lysis, rinse the UH-06 autoclave with ultrapure water, and finally store the tubing in 20% ethanol.

[0049] (2) Nickel affinity chromatography The target protein has an 8×His tag at its N-terminus, which can form a chelate with Ni ions, thereby binding to the resin column. This invention uses a GE Healthcare 5 mL HisTrap HP pre-packed column. The specific purification steps are as follows: Using a peristaltic pump, the GE Healthcare 5 mL HisTrap HP pre-packed column is washed sequentially with 10 column volumes of ultrapure water, elution buffer, and binding buffer. Then, sonication lysis buffer is loaded into the pre-packed column, and the flow-through is collected. The "ÄKTAPure" protein purification system is turned on, and the column inlet pressure, pressure differential, flow rate, and other parameters are set according to the pre-packed column parameters. Pump heads A and B are placed in the binding and elution buffers, respectively. The pre-packed column is connected to the protein purification system drop-to-drop, and the pre-packed column is washed with binding elution buffer until A... 280 Approaching baseline. Perform gradient elution with elution buffer, setting the program to "0%–100% B, 30 min". Wait for A 280 After the elution begins, start collecting the eluent, collecting 5 mL of eluted sample from each collection tube. After elution is complete, store the pre-packed column in binding buffer. According to A... 280 With A 260 Collect the corresponding collection tube for peak elution, take 10 μL, and use it for SDS-PAGE identification.

[0050] (3) Gel filtration chromatography Gel filtration chromatography separates proteins based on their molecular weight. When protein particles pass through porous gel particles, smaller proteins can enter the pores, resulting in a longer migration path and slower speed; while larger proteins cannot enter the pores and are rapidly eluted by the mobile phase. The experiment primarily used a GE Healthcare HiLoad 26 / 60 Superdex 200pg pre-packed column. The experimental procedure was as follows: The ÄKTAPure protein purification system was turned on, and the pre-packed column parameters (introduction pressure, pressure differential, flow rate, etc.) were set according to the pre-packed column parameters. The pump head (A pump) was placed in the molecular sieve buffer to equilibrate the gel column to one column volume. The protein sample was concentrated to within 10 mL using an ultrafiltration tube, transferred to a 15 mL centrifuge tube, and centrifuged at 4000 rpm, 4°C for 10 minutes to remove the precipitate. A 10 mL loop was loaded onto the ÄKTA Pure protein purifier, the loop was cleaned with a threaded syringe, and the protein sample was loaded onto the loop. The sample was then loaded onto the pre-packed column using the ÄKTA Pure protein purifier, and the collection program was started, collecting 5 mL per tube. According to A... 280 With A 260 Collect the corresponding collection tubes for peak elution, taking 10 μL for SDS-PAGE identification. Select A. 280 With A 260 Samples with correct molecular weight and high purity in the peak were concentrated to 5-10 mg / mL using an ultrafiltration tube, centrifuged at 12,000 rpm for 10 minutes at 4℃ to remove impurities, aliquoted, flash-frozen in liquid nitrogen, and stored at -80℃. 2.2.7 Complex Assembly First, the protein fractions were mixed in proportion and brought to a final volume of 500 μL with molecular sieve buffer. After incubation at 4°C for 30 minutes, the supernatant was collected by centrifugation at 12,000 rpm for 10 minutes. Using the ÄKTA Pure protein purification system, a Superose 6 Increase 10 / 300 GL column was pre-equilibrated with molecular sieve buffer (flow rate 0.5 mL / min, column inlet pressure 5 MPa, pressure differential 2.6 MPa, equilibration to one column volume). 500 μL of the complex sample was injected through a sample loop, and 0.5 mL of the elution fraction was collected from each tube. The complex binding was analyzed by SDS-PAGE, and A was selected. 280 With A 260 Samples with the correct molecular weight and high purity in the peak were concentrated to 3 mg / mL using an ultrafiltration tube and centrifuged at 12,000 rpm for 10 minutes at 4°C to remove impurities.

[0051] 2.2.8 Preparation of Frozen Samples This invention utilizes the Thermo Scientific Vitrobot Mark IV cryosample preparation system to prepare cryo-electron microscopy (cryo-EM) samples. First, the instrument chamber environment is set to 4°C and 100% humidity. New filter paper is replaced, and the chamber is equilibrated for 10 minutes. After pre-cooling the metal support and copper bowl with liquid nitrogen, ethane gas is slowly introduced to liquefy them. A porous carbon-supported gold mesh (carbon membrane side up) is placed in a culture dish and treated with glow discharge (15 mA, 60 s) to enhance its hydrophilicity. Vitrobot parameters are set (blotforce -2, blot time 3 s). 2 μL of protein sample is aspirated and added to the mesh; the instrument automatically performs aspiration filtration and rapidly immerses the sample in liquid ethane for freezing. The mesh is then transferred to liquid nitrogen and stored in the TEM sample cassette.

[0052] 2.2.9 Data Collection and Analysis of Cryo-Electron Microscopy Samples For initial screening, a cryogenic grid was mounted on a Thermo Fisher Glacios 200 kV cryo-electron microscope. After sample preparation, data acquisition was performed using the Thermo Fisher Glacios 200 kV cryo-transmission electron microscope. First, multiple fields of view were rapidly scanned in low-magnification mode to screen sample areas with moderate ice thickness (50–100 nm) and uniform protein distribution. Then, the microscope was switched to high-magnification mode, and parameters were set using automated acquisition software (such as SerialEM): electron dose 50 e. - / Ų, underfocus value -1.5 to -2.5 μm, exposure time per image 2 s, a total of 3000–5000 raw microscopic images were acquired.

[0053] The cryo-electron microscopy data processing workflow is as follows: First, motion correction is performed on the original images using MotionCor2, and the contrast transfer function (CTF) is calculated using Gctf. Then, high-quality images are manually selected, and Gautomatch is used for initial particle selection. Next, 2D classification is performed in RELION 3.0 to optimize the particle template and further extract target particles. The selected high-quality particles undergo initial 3D reconstruction in CryoSPARC, followed by 3D classification and fine-tuning to optimize the structure. Finally, atomic models are built and optimized using UCSF Chimera, Phenix, and Coot to obtain high-resolution 3D structures.

[0054] 2.2.10 Protein stability analysis Protein stability analysis was performed using a Nano Temper PR series protein stability analyzer. First, the protein sample was diluted to approximately 20 μL (0.1 mg / mL). The protein solution was then drawn into the capillary tube (PR NT.48) using siphon action. The instrument and accompanying software were turned on, the sample holder was opened, and the mirror under the sample chamber was wiped with 100% alcohol to ensure it was clean and would not interfere with capillary recognition. The capillary tube was then inserted, and the sample holder was closed. The temperature gradient mode and laser intensity were set, and protein sample A was analyzed. 350nm With A 330nm Value. Start the program and wait for the instrument to automatically collect and calculate protein T. m Values ​​were obtained, and a fitted curve was constructed. Finally, the data were analyzed using the accompanying software.

[0055] The technical solution will be clearly and completely described below through embodiments of this application. Obviously, the described embodiments are only some preferred embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of this application.

[0056] Example 1: eIF3 Mutation Design Disulfide bonds can enhance protein stability by restricting conformational freedom, thereby improving the spatial resolution of proteins in cryo-electron microscopy and aiding in structural analysis. Previous studies on the eIF3 structure revealed that the eIF3k / l subunits readily dissociate from the eIF3 core complex. Various methods can be used to reinforce the spatial structure of protein subunits; after considering multiple factors and conducting tests, adding disulfide bonds was the preferred approach. Regarding disulfide bond addition, based on the cryo-electron microscopy structure of the eIF3 core complex previously resolved in our laboratory, we designed and introduced disulfide bonds into the eIF3l, eIF3e, and eIF3f subunits using PyMol analysis. Preliminary screening yielded the following sites: eIF3e:R376C, eIF3l:S462C, eIF3e:Y401C, eIF3l:Y539C, eIF3f:S346C, and eIF3l:F550C. At the interface between eIF3e and eIF3l, the arginine at position 376 of the eIF3e subunit is located at the end of the α-helix. Mutating it to cysteine ​​will not interfere with α-helix formation, and the distance between this amino acid and the serine at position 462 of the eIF3l subunit is within 10 Å, allowing for disulfide bond formation. The other two mutations are designed near the hepta-helix bundle of the PCI domain in the eIF3 core octamer, which can resist the unfolding effect of the helix bundle under extreme conditions. The tyrosine at position 539 of the eIF3l subunit is located at the beginning of the helix bundle, close to the tyrosine at position 401 of the eIF3e subunit; the phenylalanine at position 550 of the eIF3l subunit is located in the middle of the α-helix, close to the tyrosine at position 346 of the eIF3f subunit.

[0057] Example 2: Expression of the eIF3 core octamer complex eIF3chfaem subunit prokaryotic expression and purification This invention uses the pRAP co-expression system (refer to "Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression." ACSSynthetic Biology, 2023, 12(1): 136-143) to express the human eIF3 core complex in Escherichia coli. Previous experiments revealed that co-expression of the eIF3 core octamer only resulted in the formation of the eIF3chfaem core hexamer, lacking the two eIF3kl subunits. Therefore, this invention employs a separate expression and reassembly of the eIF3chfaem hexamer and the eIF3kl dimer. The expression of the eIF3chfaem hexamer requires two plasmids: the eIF3c, h, f, and a subunits are expressed on a plasmid containing kanamycin resistance, and the eIF3e and m subunits are expressed on a plasmid containing ampicillin resistance (each subunit has a His tag at its N-terminus; the truncated form is described in the experimental materials section). These two plasmids were transformed into Hi-ControlBL21(DE3)pRARE2 E. coli competent cells and induced for expression for 18 h at low temperature (18℃) and low concentration of IPTG (0.1 mM). After high-pressure crushing and centrifugation, the plasmids were captured using a nickel affinity chromatography column. Figure 2 A), verified by SDS-PAGE ( Figure 2 (B, C) The six-subunit co-expression product was successfully obtained. Collection tubes containing the target components were collected and concentrated. Further purification was then performed using gel filtration chromatography. SDS-PAGE verification showed that the second peak represented a homogeneous protein product with all six components. Figure 2 (D, E) Approximately 10 mg of sample can be obtained per liter of bacteria. The purification methods for the three groups of different mutations in the eIF3e subunit were the same. The spectra of nickel affinity chromatography and gel filtration chromatography were similar, and the peak spectra indicated that the eIF3e point mutation did not affect the folding of the eIF3chfaem hexamer.

[0058] eIF3kl subunit prokaryotic expression and purification Co-expression of eIF3kl also requires two plasmids, eIF3k and eIF3l, on plasmids containing ampicillin resistance and kanamycin resistance, respectively (each subunit has a His tag at the N-terminus; see the Experimental Materials section for truncated forms). The expression protocol is similar to that for the expression of the eIF3chfaem hexamer. After capture using a nickel affinity chromatography column (…), Figure 3A), verified by SDS-PAGE ( Figure 3 B), successfully obtained the eIF3kl co-expression product, collected the tube containing the target component, and concentrated it. Further purification was then performed using gel filtration chromatography. SDS-PAGE verification showed that the second peak represented a homogeneous eIF3kl dimer. Figure 3 (C, D) Approximately 1.5 mg of sample can be obtained per liter of bacteria. The purification methods for the three groups of different mutations in the eIF3l subunit were the same. The spectra of nickel affinity chromatography and gel filtration chromatography were similar, and the peak spectra showed that the point mutation of eIF3l did not affect the folding of eIF3kl dimer.

[0059] eIF3 core octomer assembly Assembly of the eIF3 core octomer requires mixing the eIF3chfaem hexamer and the eIF3kl dimer at a molar ratio of 1:2. Three mutant groups are prepared, incubated at low temperature (4°C) for 30 minutes, and then further purified using a gel filtration chromatography column. Figure 4 (A, C, E). SDS-PAGE analysis revealed that the first peak represented the homogeneous eIF3 core octamer, while the second peak primarily represented the excess eIF3kl dimer in the sample. After assembly of the three eIF3 mutants, they were filtered through the same HiLoad 16 / 60 Superdex 75pg gel filtration column. The peak positions and shapes did not appear to affect the overall folding of the eIF3 core complex; further biochemical and structural analysis is needed to validate the complex.

[0060] Stability verification of eIF3 mutant After co-incubating eIF3chfaem with eIF3kl containing a single cysteine ​​mutation for 30 minutes, disulfide bonds were theoretically possible to form due to the spatial proximity of the two cysteine ​​residues in the three mutant groups. Non-reducing SDS-PAGE analysis of the core octamer of eIF3 containing the cysteine ​​mutation revealed that all three mutant groups were able to form disulfide bonds. Figure 5 The first group of mutations can form disulfide bonds well between eIF3l and eIF3e, while the second and third groups of mutations form disulfide bonds with the same subunit of eIF3l, but fail to form disulfide bonds between eIF3l and different subunits of eIF3f and e. The disulfide bonds formed by the first group of mutations enhance the interaction strength between eIF3kl and eIF3chfaem, and also promote the stability of eIF3 to some extent.

[0061] The paper utilizes the UV absorbance ratio (A) caused by monitoring changes in the microenvironment of tryptophan residues. 350nm / A 330nm The melting temperature (T) varies with temperature, and its melting temperature is calculated. mThis method determines the folding pattern of mutants. It is based on the fact that during protein denaturation, tryptophan undergoes a characteristic red shift in its UV absorption spectrum when it is exposed from the hydrophobic core to the hydrophilic surface. For example... Figure 6 As shown in C, A 350nm / A 330nm The ratio of eIF3e to eIF3l exhibits a typical S-shaped curve increase with temperature. By fitting the curve using the Boltzmann equation, the Tf ratio between the wild type and the three mutant groups (in the following order: eIF3e:R376C, eIF3l:S462C; eIF3e:Y401C, eIF3l:Y539C; eIF3f:S346C, eIF3l:F550C) was obtained. m The values ​​were 49.25℃, 49.48℃, 48.78℃, and 49.43℃, respectively. The three groups of mutant T... m The value is similar to that of the wild-type eIF3 core complex, which indirectly reflects that the mutation did not affect the folding state of the eIF3 core complex.

[0062] Example 3: Data Collection and Analysis of eIF3 Core Complex and eIF4G Assembled Samples eIF3 core complex sample Cryo-electron microscopy images of the three mutant eIF3 core octamer and the natural eIF3 core octamer are shown below. Figure 7 In the field of view, the eIF3 core complex particles with the eIF3e:R376C and eIF3l:S462C mutations were uniform in size and distribution. Figure 7 B). The natural eIF3 core octamer contains a large number of dispersed subunits ( Figure 7 A). Numerous dispersed eIF3 subunits were also observed in the eIF3 core octamer of mutants carrying eIF3e:Y401C, eIF3l:Y539C; eIF3f:S346C, and eIF3l:F550C, respectively. Figure 7 (C, D). This indicates that the eIF3 core complex with the eIF3e:R376C and eIF3l:S462C mutations can better maintain the stability of eIF3. Since the other two mutations failed to improve the stability of eIF3 in cryo-electron microscopy images, they were not used in subsequent studies.

[0063] Two-dimensional classification results further illustrate that the eIF3 core complex with the eIF3e:R376C and eIF3l:S462C mutations is more stable than the native eIF3 core complex. The native eIF3 core octamer contains a large number of eIF3 subunit images (…). Figure 8 A), while the eIF3 core octamer with the eIF3e:R376C and eIF3l:S462C mutations is mostly a complete eIF3 "five-lobed" structure ( Figure 8B). Given that the eIF3 core octamer with the eIF3e:R376C, eIF3l:S462C mutations is in good condition under cryo-electron microscopy, it is suitable for subsequent assembly with the eIF4g, initiating complex.

[0064] For electron microscopy images of eIF3 core octamers with the eIF3e:R376C and eIF3l:S462C mutations, 903,279 particles were automatically selected and extracted using RELLION 3.0. After multiple rounds of 2D and 3D classification, 704,239 good particles were identified. Following several rounds of 3D classification optimization, four types of relatively good eIF3 core complexes were obtained. These particles were then imported into CryoSPARC software for 3D reconstruction, yielding eIF3 core complexes with a resolution of 3.14 Å. Figure 9 ).

[0065] eIF3 core complex and eIF4G assembly sample Given the favorable performance of the eIF3 core octamer with the mutations eIF3e:R376C and eIF3l:S462C, the eIF3 core complex was assembled with eIF4G to determine its functional activity. During assembly, a large amount of white flocculent precipitate appeared when the eIF3 core complex was incubated with the eIF4G sample. The precipitate dissolved after the addition of a small amount of 1 M NH4Cl, indicating that the mutated eIF3 core complex may interact with eIF4G. Therefore, cryo-electron microscopy was directly performed for sample preparation and data collection. The two-dimensional classification results of the eIF3 core complex and eIF4G cryo-electron microscopy are shown below. Figure 10 The size of the eIF3 core complex and the eIF4G assembled particles can be observed ( Figure 10 Compared to the eIF3 core complex ( Figure 8 Regarding B), there is a slight signal between the a and c subunits ( Figure 10 (Red box). This indicates that the mutated eIF3 core octamer has good functional activity.

Claims

1. A method for preparing an eIF3 octamer mutant, characterized in that, Obtain a polypeptide with a mutated eIF3 core subunit, including but not limited to eIF3a, eIF3c, eIF3e, eIF3k, eIF3l, eIF3m, eIF3f, and eIF3h; the polypeptide folds to form subunits and then assembles into an eIF3 core octamer; the mutation is selected from one or more of the following sites: eIF3e:R376C, eIF3l:S462C; eIF3e:Y401C, eIF3l:Y539C; eIF3f:S346C, eIF3l:F550C.

2. The preparation method according to claim 1, characterized in that, Obtaining peptides with mutated eIF3 core subunits, including expressing nucleic acids encoding mutated eIF3 core subunits as corresponding proteins; Assemble into an eIF3 core octamer by mixing the protein components of the eIF3 core subunit in a specific ratio, incubating on ice, and then collecting the eIF3 core octamer; the molar number of eIF3c / h / f / a / e / m subunits is the same, the molar number of eIF3k / l subunits is the same, and the molar ratio of eIF3c to eIF3k subunits is 1:(1-3).

3. The preparation method according to claim 2, characterized in that, The method for preparing the nucleic acid encoding the mutated eIF3 core subunit is as follows: using the nucleic acid encoding the eIF3 core subunit as an amplification template, the mutation site is designed in the center of the complementary primer, with 5-10 bases extending on each side to ensure that there are 15-20 bases paired between the two primers, and each primer is further extended by 5-10 bases at the 5' end to ensure primer specificity. Plasmids encoding nucleic acids containing the eIF3 core subunit were amplified using polymerase chain reaction.

4. The preparation method according to claim 3, characterized in that, The amplification system for polymerase chain reaction is shown in Table 9, the amplification reaction procedure is shown in Table 10, or the amplification primers are shown in Table 1.

5. The preparation method according to claim 2, characterized in that, The protein components of the eIF3 core subunit are mixed in a certain proportion. This can be done by first obtaining an equimolar amount of eIF3chfaem hexamer, and then mixing it with the eIF3kl subunit; or by directly mixing the individual core octamer subunits. The number of moles of eIF3c / h / f / a / e / m subunits is the same, and the number of moles of eIF3k / l subunits is the same. The molar ratio of eIF3c to eIF3k subunits is 1:(1.1-2).

6. The preparation method according to claim 1, characterized in that, The preparation method includes protein purification, which includes one or more steps such as high-pressure crushing, nickel affinity chromatography, and gel filtration chromatography. High-pressure disruption step: Resuspend the bacterial culture overexpressing the eIF3 core subunit using binding buffer, add protease inhibitor cocktail and PMSF solution, mix well, and then use a high-pressure cell disruptor to pressurize to 800 bar to disrupt the bacterial cells. Stop pressurizing when the color of the solution at the outlet changes from off-white to transparent, and collect the lysate; centrifuge at a speed of not less than 12,000 rpm at 0-4℃ for not less than 40 minutes, and collect the lysate supernatant; Nickel affinity chromatography: The pyrolysis solution containing the eIF3 core subunit was loaded into a pre-packed column and the flow-through was collected; Turn on the protein purification system and connect the pre-packed column dropwise. Wash the pre-packed column with binding elution buffer until A... 280 Close to the baseline; Perform gradient elution with elution buffer and wait for A 280 Once the eluent begins to rise, begin collecting the eluent. Gel filtration chromatography: The lysate containing the eIF3 core subunit was concentrated to within 10 mL using an ultrafiltration tube, and the precipitate was removed by centrifugation at 3000-6000 rpm and 0-4℃ for 5-20 minutes. Load the loop into the protein purifier, clean the loop with a threaded syringe, and load the protein sample containing the eIF3 core subunit into the loop; use the protein purifier to load the protein sample onto the pre-packed column and begin the collection procedure.

7. An eIF3 octamer mutant, characterized in that, It contains one or more of the following mutations: eIF3e:R376C, eIF3l:S462C; eIF3e:Y401C, eIF3l:Y539C; or eIF3f: S346C, eIF3l:F550C.

8. The eIF3 octamer mutant according to claim 7, characterized in that, Obtained according to the preparation method described in any one of claims 1-6.

9. The application of the preparation method according to any one of claims 1-6 improves the stability of eIF3 octamer.

10. An mRNA translation kit, characterized in that, The mRNA translation kit contains the eIF3 octamer mutant as described in claim 7 or 8.

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