Cell lysis supernatant as well as preparation method and application thereof

Abstract

The invention relates to the field of molecular biology, and particularly discloses a cell lysis supernatant as well as a preparation method and application thereof. Wherein the cell lysis supernatant comprises an eIF2alpha mutant and a GADD34 truncated body; the eIF2alpha mutant is obtained by mutating the 52nd amino acid of eIF2alpha, and the GADD34 truncated body is obtained by knocking out the 1st to 240th amino acids of GADD34. Compared with a normal cell lysis supernatant, the cell lysis supernatant provided by the invention mutates the phosphorylated translation initiation factor to avoid phosphorylation, and can dephosphorylate the phosphorylated translation initiation factor to ensure that the cell lysis supernatant has high translation activity.

Description

Technical Field

[0001] The present application relates to the field of molecular biology technology, and in particular to a cell lysis supernatant and a preparation method and application thereof. Background Art

[0002] Cell-free protein expression is a technology for expressing proteins in vitro. It uses mRNA as a template and forms a protein in vitro translation system by adding cell lysis supernatant and other necessary substances to complete protein translation and achieve protein expression. In the absence of conflicts in intrinsic cellular metabolism or biochemical pathways, membrane proteins, viral proteins, toxic proteins, and proteins that undergo rapid proteolysis through intracellular processes are more easily expressed. Therefore, cell-free protein expression can be used as an ideal method for synthesizing proteins that are difficult to express and subsequently analyze using traditional methods.

[0003] In the process of making cell lysis supernatant, cells are inevitably physically broken or chemically lysed, etc. This process will cause stress response in cells. One of the reactions that affect protein translation in stress response is eIF2α phosphorylation. Phosphorylated eIF2α occupies the site where eIF2B interacts with normal eIF2α, blocking the exchange of GDP and GTP mediated by eIF2B, preventing the formation of translation initiation complex, and reducing the translation activity of cell lysis supernatant. Therefore, reducing or avoiding the phosphorylation reaction of eIF2α in the preparation of cell lysis supernatant is an urgent problem to be solved. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a cell lysis supernatant and a preparation method and application thereof, aiming to solve the problem of reduced translation activity of the cell lysis supernatant caused by eIF2α phosphorylation during the preparation of the cell lysis supernatant.

[0005] The technical solution of this application is as follows:

[0006] In a first aspect of the present application, a cell lysis supernatant is provided, comprising an eIF2α mutant and a GADD34 truncate; the eIF2α mutant is obtained by mutating the amino acid at position 52 of eIF2α, and the GADD34 truncate is obtained by knocking out the amino acids at positions 1-240 of GADD34.

[0007] Optionally, the amino acid sequence of the eIF2α mutant is shown as SEQ ID NO.1, and the nucleic acid sequence of the gene encoding the eIF2α mutant is shown as SEQ ID NO.2.

[0008] Optionally, the amino acid sequence of the GADD34 truncation is shown as SEQ ID NO.3, and the nucleic acid sequence of the gene encoding the GADD34 truncation is shown as SEQ ID NO.4.

[0009] The second aspect of the present application provides a method for preparing the cell lysis supernatant of the first aspect of the present application, comprising the steps of obtaining cells transiently co-expressing an eIF2α mutant and a GADD34 truncate, resuspending the cells by adding a buffer, disrupting the cells in an ice bath environment, centrifuging to precipitate the cell fragments, and obtaining the cell lysis supernatant.

[0010] Optionally, the method for preparing cells that transiently co-express eIF2α mutant and GADD34 truncation comprises the steps of: obtaining a recombinant expression plasmid of GADD34 truncation and a recombinant expression plasmid of eIF2α mutant; and transfecting the recombinant expression plasmid of GADD34 truncation and the recombinant expression plasmid of eIF2α mutant into the same mammalian cell.

[0011] Optionally, the method for preparing the recombinant expression plasmid of the eIF2α mutant includes the steps of: using a gene fragment containing a nucleic acid sequence as shown in SEQ ID NO.5 as a template, performing PCR amplification using a first primer pair to obtain the coding gene of the eIF2α mutant; transferring the coding gene of the eIF2α mutant into a plasmid to obtain the recombinant expression plasmid of the eIF2α mutant.

[0012] Optionally, the nucleic acid sequence of the first primer pair is shown as SEQ ID NO.6 and SEQ ID NO.7.

[0013] Optionally, the method for preparing the recombinant expression plasmid of the GADD34 truncation includes the steps of: using the cDNA library of the wild-type HeLa cell line as a template, performing PCR amplification using a second primer pair to obtain the coding gene of the GADD34 truncation; transferring the coding gene of the GADD34 truncation into a plasmid to obtain the recombinant expression plasmid of the GADD34 truncation.

[0014] Optionally, the nucleic acid sequence of the second primer pair is shown as SEQ ID NO.8 and SEQ ID NO.9.

[0015] The third aspect of the present application provides a use of the cell lysis supernatant of the first aspect of the present application in cell-free expression.

[0016] Beneficial effects of the present application: the cell lysis supernatant of the present application includes an eIF2α mutant and a GADD34 truncate, wherein the eIF2α mutant is obtained by mutating the amino acid at position 52 of eIF2α, and compared with eIF2α, the eIF2α mutant will not be affected by phosphorylation, and thus will not reduce the translation activity of the cell lysis supernatant; the GADD34 truncate is obtained by knocking out the amino acids at positions 1-240 of GADD34, and the half-life of the GADD34 truncate is longer than that of GADD34, so that the GADD34 truncate can prolong the effect of dephosphorylating the phosphorylated translation initiation factor, thereby improving the translation activity of the cell lysis supernatant. That is, compared with the normal cell lysis supernatant, the cell lysis supernatant including the eIF2α mutant and the GADD34 truncate mutates the phosphorylated translation initiation factor and dephosphorylates the phosphorylated translation initiation factor, thereby ensuring that the cell lysis supernatant has a higher translation activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for describing the embodiments are briefly introduced below.

[0018] Figure 1 This is a graph showing the activity of luciferase produced by in vitro translation in Example 2 provided in the present application;

[0019] Figure 2 This is an SDS-PAGE analysis diagram of the recombinant protein produced by in vitro translation in Example 3 provided in the examples of the present application;

[0020] Figure 3 This is a graph showing the activity of the nano-luciferase produced by in vitro translation in Example 4 provided in the present application;

[0021] Figure 4 This is a graph for detecting the activity of luciferase produced by in vitro translation in Example 5 provided in the examples of the present application. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0023] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in the field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0024] The present application provides a cell lysis supernatant, including an eIF2α mutant (eIF2α(S52A)) and a GADD34 truncate (GADD34Δ240). The eIF2α mutant is obtained by mutating the amino acid at position 52 of eIF2α, and the GADD34 truncate is obtained by knocking out the amino acids at positions 1-240 of GADD34.

[0025] The translation process includes three stages: initiation, translation, and termination. In the initiation stage, the eIF2 initiation complex integrates various stress-related signals to regulate global and specific mRNA translation. Under appropriate conditions, eIF2 binds to GTP and Met-tRNAi to form a ternary complex (TC), which then binds to the 40S ribosomal subunit, eIF1, eIF1A, eIF5, and eIF3 to form the 43S pre-initiation complex (PIC). The 43S PIC scans the mRNA UTR for the AUG start codon. After recognizing AUG, eIF2 can hydrolyze GTP to GDP with the help of the GTPase activating protein eIF5 and decompose it from the mRNA to bind to the 60S ribosomal subunit and extend the polypeptide chain. In the presence of eIF5 as GDI, eIF2 remains bound to GDP. In order to start another round of translation, eIF2B must act as both a GDI displacement factor (GDF) and a guanine exchange factor (GEF) to exchange GDP for GTP on eIF2. This step must be closely regulated. After phosphorylation of eIF2α by four stress-activated kinases from different families (PKR (dsRNA), PERK (ER stress), GCN2 (amino acid starvation), and HRI (heme deficiency)), eIF2 can act as a dominant negative complex to sequester eIF2B, thereby blocking nucleotide exchange and reducing translation activity. eIF2α, eIF2β, and eIF2γ together form the eIF2 complex, of which eIF2α is the main regulatory subunit, containing phosphorylation and RNA binding sites.

[0026] The 52nd amino acid of eIF2α is a common phosphorylation site for the above four kinases. Therefore, by mutating the 52nd amino acid of eIF2α, the eIF2α mutant can function normally and is not affected by phosphorylation, thereby improving the translation activity of the cell lysate supernatant.

[0027] GADD34 can recruit the phosphatase PP1c to eIF2, thereby mediating the dephosphorylation of eIF2α. The C-terminus of GADD34 contains a PEST repeat sequence and a KVRF matrix sequence, both of which are important for binding to PP1c and subsequently dephosphorylating eIF2α. The N-terminal region of GADD34 includes a membrane binding or targeting region (MEMB, amino acids 1-180). MEMB localizes GADD34 to the endoplasmic reticulum of mammalian cells and is also the reason why GADD34 is rapidly degraded by the 26S proteasome in mammalian cells. The N-terminal amino acids 1-240 of GADD34 were knocked out, removing the membrane binding region of GADD34, thereby making the GADD34 truncate less susceptible to rapid degradation, and can continuously dephosphorylate phosphorylated translation initiation factors, thereby increasing the translation activity of the cell lysate supernatant.

[0028] The cell lysis supernatant in the present application includes eIF2α mutants and GADD34 truncations, which solve the problem of eIF2α phosphorylation in the preparation of cell lysis supernatant from two aspects: phosphorylation of eIF2 and dephosphorylation of phosphorylated eIF2, so that the cell lysis supernatant has higher translation activity.

[0029] In some embodiments, the amino acid sequence of the eIF2α mutant is shown as SEQ ID NO.1, and the nucleic acid sequence of the gene encoding the eIF2α mutant is shown as SEQ ID NO.2.

[0030] In some embodiments, the amino acid sequence of the GADD34 truncation is shown as SEQ ID NO.3, and the nucleic acid sequence of the gene encoding the GADD34 truncation is shown as SEQ ID NO.4.

[0031] The present application also provides a method for preparing the cell lysis supernatant as described above in the present application, comprising the following steps:

[0032] Cells transiently co-expressing eIF2α mutants and GADD34 truncations were obtained, and buffer was added to resuspend the cells. The cells were disrupted in an ice bath environment, and centrifuged to precipitate the cell fragments to obtain a cell lysis supernatant.

[0033] In some embodiments, the volume ratio of cells transiently co-expressing eIF2α mutant and GADD34 truncation to buffer is 1:2. The culture fluid of the cultured cells is removed by centrifugation, and the cells are washed several times with buffer and centrifuged to remove the buffer to obtain cells transiently co-expressing eIF2α mutant and GADD34 truncation. The buffer is HEPES buffer.

[0034] Furthermore, a method for preparing cells transiently co-expressing an eIF2α mutant and a GADD34 truncate comprises the steps of:

[0035] S1. Obtain the recombinant expression plasmid of GADD34 truncation and the recombinant expression plasmid of eIF2α mutant.

[0036] S2. Transfect the recombinant expression plasmid of GADD34 truncation and the recombinant expression plasmid of eIF2α mutant into the same mammalian cell to obtain the result. The mammalian cell can be HEK293F cell.

[0037] In some embodiments, the method for preparing a recombinant expression plasmid of an eIF2α mutant comprises the steps of:

[0038] S111, using the gene fragment containing the nucleic acid sequence shown in SEQ ID NO.5 as a template, using the first primer pair to perform PCR amplification to obtain the coding gene of the eIF2α mutant.

[0039] S112, transferring the coding gene of the eIF2α mutant into a plasmid to obtain a recombinant expression plasmid of the eIF2α mutant.

[0040] The gene fragment containing the nucleic acid sequence shown in SEQ ID NO.5 can be a linear DNA fragment or a plasmid containing the gene encoding eIF2α. The plasmid used to transfer the gene encoding the eIF2α mutant into the plasmid is selected from pMlink or pCMV.

[0041] In some embodiments, the nucleic acid sequence of the first primer pair is shown as SEQ ID NO.6 and SEQ ID NO.7.

[0042] In some embodiments, the method for preparing a recombinant expression plasmid of GADD34 truncation comprises the steps of:

[0043] S121. Using the cDNA library of the wild-type HeLa cell line as a template, PCR amplification was performed using the second primer pair to obtain the coding gene of the GADD34 truncation.

[0044] S122. Transfer the coding gene of the GADD34 truncation into a plasmid to obtain a recombinant expression plasmid of the GADD34 truncation.

[0045] The plasmid used to transfer the coding gene of GADD34 truncation into the plasmid is selected from pMlink or pCMV.

[0046] In some embodiments, the nucleic acid sequence of the second primer pair is shown as SEQ ID NO.8 and SEQ ID NO.9.

[0047] The present application also provides an application of the cell lysis supernatant as described above in the present application embodiment in cell-free expression.

[0048] The following is further described by means of specific examples.

[0049] Experimental materials and reagents:

[0050] Escherichia coli DH5α strain, eukaryotic expression vector pMLink, human HEK293F cells; restriction endonucleases, T4 DNA ligase and corresponding buffers were purchased from NEB; 2×HyperFusion TM High-Fidelity MasterMix (with dye) was purchased from APExBIO; HEPES buffer, sodium salt, potassium acetate, magnesium acetate, DTT, ampicillin sodium, yeast extract, and trypsin were purchased from Sangon Biotech (Shanghai) Co., Ltd.; agarose and transfection reagents PEI Transfection Reagent was purchased from Beijing Quanshijin Company; primer synthesis and gene sequencing were completed by Beijing Qingke Xinye Biotechnology Co., Ltd.; Escherichia coli culture medium was LB medium; HEK293F culture medium was Union293 cell culture medium purchased from Yonglian Biotechnology (Shanghai) Co., Ltd.

[0051] Unless otherwise specified, the experimental methods in the examples can refer to the instructions of the reagents and are conventional experimental methods in the art.

[0052] Example 1

[0053] (1) Construction of a recombinant expression plasmid of eIF2α (S52A). PCR amplification was performed using a primer pair (eIF2α-S52A-F and eIF2α-S52A-R) with nucleic acid sequences as shown in SEQ ID NO. 6 and SEQ ID NO. 7, with the plasmid containing the eIF2α coding gene as a template. The PCR reaction system and reaction procedure are shown in Tables 1 and 2.

[0054] Table 1 PCR reaction system

[0055]

[0056] Table 2 PCR reaction program

[0057]

[0058] The obtained PCR product and pMLink plasmid were double-digested with KpnI and BamHI. The enzyme digestion system is shown in Table 3.

[0059] Table 3 Enzyme digestion reaction system

[0060]

[0061] The two enzyme-digested products were ligated using T4 DNA ligase. The ligation system is shown in Table 4.

[0062] Table 4 Ligation reaction system

[0063]

[0064]

[0065] The ligation product was transferred into DH5α competent cells, and the cells were plated on LB culture plates for overnight culture. Single colonies were picked from the plates and sent to Sanger sequencing to verify the sequencing results to obtain monoclonal colonies of the plasmid recombinantly expressing eIF2α (S52A), which were then cultured, amplified and extracted to obtain the recombinant expression plasmid of eIF2α (S52A).

[0066] (2) Construct a recombinant expression plasmid of GADD34Δ240. Use the primer pair (GADD34Δ240-F and GADD34Δ240-R) with nucleic acid sequences as shown in SEQ ID NO.8 and SEQ ID NO.9, and perform PCR amplification with the cDNA library of wild-type Hela cells as a template; perform double restriction digestion of the obtained PCR product and pMLink plasmid with KpnI and BamHI; connect the two restriction products using T4 DNA ligase; transfer the connection product into DH5α competent cells, and spread the cells on LB culture plates for overnight culture. Pick a single colony from the plate and send it to Sanger sequencing to verify the sequencing results to obtain a single clone colony of the plasmid for recombinant expression of GADD34Δ240, and then perform culture amplification and extraction to obtain the recombinant expression plasmid of GADD34Δ240. The PCR reaction system and reaction procedure, double restriction digestion reaction system, and connection reaction system are similar to those in (1) and will not be repeated.

[0067] (3) Preparation of cells transiently co-expressing eIF2α(S52A) and GADD34Δ240.

[0068] After HEK-293 cells were revived, they were cultured in a 37°C, 5% CO2 constant temperature shaker and passaged for more than 3 times to restore the cell state. 5 cells / mL and continue culturing. When the cells are in the logarithmic growth phase (generally 2-3×10 6 cells / mL) and the cell viability is greater than 98%, start transfection. DNA dilution tube: add 45 μg of the plasmid prepared in (1) and the plasmid prepared in (2) to 2.5 mL PEI Buffer and mix gently. PEI dilution tube: add 270 μL PEI to this 2.5 mL PEI Buffer and mix gently. Add all the liquid in the PEI dilution tube to the DNA dilution tube, gently blow and mix, and refine at room temperature for 10-15 minutes to obtain a transfection complex. Add the transfection complex to the cells and culture at 37°C to obtain cells that transiently co-express eIF2α (S52A) and GADD34Δ240.

[0069] (4) Prepare cell lysis supernatant.

[0070] The cells in (3) were collected by centrifugation, washed with buffer, resuspended in HEPES buffer at a volume ratio of 1:2 of HEPES buffer to cells according to the fresh weight of the cells, and disrupted by using a Dounce tissue grinder in an ice bath environment, and the cell disrupted material was precipitated in a high-speed centrifuge at 4°C to obtain a cell lysis supernatant.

[0071] Example 2

[0072] In this example, luciferase (Firefly Luciferase) was translated in vitro, and the concentration and activity of the product were detected. The relevant buffer (6× reaction buffer, Creatine phosphokinase) and luciferase substrate (Firefly Luciferase substrate) were from Promega's ONE-Glo TM Luciferase Assay System.

[0073] According to the reaction system shown in Table 5, the reaction was carried out at 37° C. for 1 hour to synthesize luciferase (Firefly luciferase). The experimental group was added with the cell lysis supernatant prepared in Example 1 (containing the GADD34 truncated eIF2α mutant), and the control group was added with normal cell lysis supernatant.

[0074] Table 5 Synthesis reaction system of luciferase

[0075]

[0076] An equal volume (30uL) of Firefly Luciferase substrate was added to the reaction products of each group. After reacting for 2 minutes, the fluorescence value was detected using a multifunctional plate reader (enzyme reader, grating type EnSpire), and the detected fluorescence value was used to represent the luciferase activity. Figure 1 As shown, Figure 1 ctrl represents the control group, and eIF2α(S52A)+GADD34Δ240 represents the experimental group.

[0077] from Figure 1 As can be seen, the activity value of the product in the eIF2α(S52A)+GADD34Δ240 group was significantly higher than that in the control group, which further indicated that the cell lysis supernatant containing eIF2α(S52A) and GADD34Δ240 had good translation activity, and was significantly higher than that of the cell lysis supernatant containing eIF2α(S52A).

[0078] Example 3

[0079] In this example, the recombinant protein was translated in vitro and its concentration was detected. The recombinant protein was a protein with a size of about 20 kDa and a FLAG-tag. The relevant buffer was the same as that in Example 2.

[0080] The recombinant protein was synthesized by in vitro translation at 37°C for 1 hour in a reaction system similar to that in Example 2. The experimental group was added with the cell lysis supernatant prepared in Example 1 (containing GADD34 truncation and eIF2α mutant), and the control group was added with normal cell lysis supernatant.

[0081] The reaction products of each group were analyzed by Western Blot using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 2 As shown, Figure 2 The middle maker lane represents protein Marker, the CRTL lane represents the control group, and the eIF2α(S52A)+GADD34Δ240 lane represents the experimental group.

[0082] from Figure 2It can be seen that a relatively obvious band of about 20 kDa appeared in the lane of the experimental group, and a band of about 20 kDa could be vaguely seen in the control group. In the experimental group, the band in the lane of eIF2α(S52A)+GADD34Δ240 was the most obvious, indicating that the content of the recombinant protein obtained by in vitro translation in the eIF2α(S52A)+GADD34Δ240 group was the highest, which further indicated that the cell lysis supernatant containing eIF2α(S52A) and GADD34Δ240 had good translation activity, and was significantly higher than that of the cell lysis supernatant containing eIF2α(S52A).

[0083] Example 4

[0084] In this example, Nano Luciferase was translated in vitro and the concentration and activity of the product were detected. The relevant buffer was the same as that in Example 2.

[0085] Nano-luciferase was synthesized by reacting at 37° C. for 1 h in a similar reaction system as in Example 2. The experimental group was added with the cell lysis supernatant prepared in Example 1 (containing GADD34 truncation and eIF2α mutant), and the control group was added with normal cell lysis supernatant.

[0086] An equal volume of 30uL NanoLuc Luciferase substrate was added to the reaction products of each group. After reacting for 2 minutes, the fluorescence value was detected using a multifunctional plate reader (enzyme reader, grating type EnSpire). The detected fluorescence value was used to represent the luciferase activity. The results are shown in Figure 3 As shown, Figure 3 ctrl represents the control group, and eIF2α(S52A)+GADD34Δ240 represents the experimental group.

[0087] from Figure 3 It can be seen that the activity value of the product in the eIF2α(S52A)+GADD34Δ240 group is significantly higher than that in the control group, indicating that the luciferase content in this group is higher, and further indicating that the cell lysis supernatant containing eIF2α(S52A) and GADD34Δ240 has good translation activity, and is significantly higher than the cell lysis supernatant containing eIF2α(S52A).

[0088] Example 5

[0089] In this example, cap-dependent translation initiation luciferase was translated in vitro at different temperatures and the activity was measured. The relevant buffer was the same as in Example 2.

[0090] According to a reaction system similar to Example 2, at 25°C, 27.5°C, 30°C, 33.5°C, 37°C and 42°C, the cell lysis supernatant prepared in Example 1 (containing GADD34 truncation and eIF2α mutant), the cell lysis supernatant prepared from cells overexpressing eIF2α mutant, the cell lysis supernatant prepared from cells overexpressing GADD34 truncation and the normal cell lysis supernatant were used as cell extracts, and the luciferase mRNA with cap-dependent translation initiation in the reaction system was translated in vitro. An equal volume (30uL) of Firefly Luciferase substrate was added to the products obtained by the reaction, and the fluorescence value was detected using a multifunctional plate reader (microplate reader, grating type EnSpire) after 2 minutes of reaction. The detected fluorescence value is used to represent the luciferase activity. The results are shown in FIG. Figure 4 As shown, Figure 4 ctrl represents the control group, and eIF2α(S52A)+GADD34Δ240 represents the experimental group. Figure 4 It can be seen that the activity of luciferase is quite different at 27.5℃ and 33.5℃, indicating that this method can maximize the efficiency of in vitro translation reaction at 27.5℃ and 33.5℃.

[0091] In summary, the cell lysis supernatant of the present application includes eIF2α mutants and GADD34 truncations. Compared with normal cell lysis supernatants, the phosphorylated translation initiation factors are mutated and dephosphorylated, ensuring higher translation activity. The cell lysis supernatant of the present application is used for cell-free expression of multiple proteins.

[0092] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.

Claims

1. A cell lysis supernatant, characterized in that: It includes eIF2α mutant and GADD34 truncation; the eIF2α mutant is obtained by mutating the 52nd amino acid of eIF2α, and the GADD34 truncation is obtained by knocking out the 1st to 240th amino acids of GADD34.

2. The cell lysis supernatant according to claim 1, characterized in that The amino acid sequence of the eIF2α mutant is shown in SEQ ID NO.1, and the nucleic acid sequence of the gene encoding the eIF2α mutant is shown in SEQ ID NO.

2.

3. The cell lysis supernatant according to claim 1, characterized in that The amino acid sequence of the GADD34 truncation is shown in SEQ ID NO.3, and the nucleic acid sequence of the gene encoding the GADD34 truncation is shown in SEQ ID NO.

4.

4. A method for preparing the cell lysis supernatant according to any one of claims 1 to 3, characterized in that: Includes steps: The cells transiently co-expressing the eIF2α mutant and the GADD34 truncate are obtained, and a buffer is added to resuspend the cells, and the cells are disrupted in an ice bath environment, and centrifuged to precipitate the cell disrupted material to obtain the cell lysis supernatant.

5. The method for preparing cell lysis supernatant according to claim 4, characterized in that: The method for preparing cells transiently co-expressing eIF2α mutant and GADD34 truncation comprises the steps of: Obtaining the recombinant expression plasmids of GADD34 truncation and eIF2α mutant; The recombinant expression plasmid of GADD34 truncation and the recombinant expression plasmid of eIF2α mutant were transfected into the same mammalian cell to obtain the result.

6. The method for preparing cell lysis supernatant according to claim 5, characterized in that: The method for preparing the recombinant expression plasmid of the eIF2α mutant comprises the steps of: Using a gene fragment containing the nucleic acid sequence shown in SEQ ID NO.5 as a template, PCR amplification is performed using a first primer pair to obtain the coding gene of the eIF2α mutant; The coding gene of the eIF2α mutant is transferred into a plasmid to obtain a recombinant expression plasmid of the eIF2α mutant.

7. The method for preparing cell lysis supernatant according to claim 6, characterized in that: The nucleic acid sequences of the first primer pair are shown as SEQ ID NO.6 and SEQ ID NO.

7.

8. The method for preparing cell lysis supernatant according to claim 5, characterized in that: The method for preparing the recombinant expression plasmid of the GADD34 truncation comprises the following steps: Using a cDNA library of a wild-type HeLa cell line as a template, performing PCR amplification using a second primer pair to obtain the coding gene of the GADD34 truncation; The coding gene of the GADD34 truncation is transferred into a plasmid to obtain a recombinant expression plasmid of the GADD34 truncation.

9. The method for preparing cell lysis supernatant according to claim 8, characterized in that: The nucleic acid sequences of the second primer pair are shown as SEQ ID NO.8 and SEQ ID NO.

9.

10. Use of the cell lysis supernatant according to any one of claims 1 to 3 in cell-free expression.

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