Recombinant hLIF mutant with improved thermal stability and preparation method and application thereof

Abstract

The present application discloses a recombinant hLIF mutant with improved thermal stability, its preparation method and application, and belongs to the field of biomedicine technology. The recombinant hLIF mutant is a point mutation that occurs on wild-type hLIF based on energy and evolutionary methods, including one or more point mutations in which the threonine at position 65 of the wild-type hLIF is mutated to glutamine (T65Q), the alanine at position 119 is mutated to leucine (A119L), and the serine at position 127 is mutated to isoleucine (S127I). The recombinant hLIF mutant has biological activity, improved thermal stability and a longer biological activity half-life. At the same time, the recombinant hLIF mutant is fused with TrxA to prepare a recombinant TrxA-hLIF mutant with improved thermal stability and biological activity, which can be used in the fields of cell biology research and medicine.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a recombinant hLIF mutant with improved thermal stability, a preparation method, and an application thereof. Background Art

[0002] Leukemia Inhibitory Factor (LIF) is a pleiotropic cytokine belonging to the IL-6 subfamily. LIF can bind to the IL-6 subfamily's co-receptor gp130 and then to its own receptor, the LIF receptor, to form a ternary complex. The functions of LIF include promoting the proliferation of primordial germ cells, regulating blastocyst implantation and early pregnancy, and maintaining the pluripotency of embryonic stem cells. LIF is a 180-amino acid polypeptide with a core protein molecular weight of 20 kDa. It has seven glycosylation sites and six cysteines. Intramolecular disulfide bonds may play an important role in maintaining the structure and biological activity of the LIF molecule. Human LIF (hLIF) shares high homology with LIF from other animal sources, such as mouse and porcine, at the amino acid level, and hLIF exhibits some activity against cells from both mouse and porcine cells. However, wild-type LIF has low thermal stability and a short half-life, making it unsuitable for culturing a variety of cells, including stem cells (SC). Therefore, improving the thermodynamic stability and biological activity half-life of LIF is particularly important in applications such as in vitro cell culture.

[0003] Existing LIF production typically involves expressing recombinant LIF in E. coli. However, LIF contains three pairs of disulfide bonds, making it prone to forming inclusion bodies when expressed in E. coli. Therefore, thioredoxin (TrxA) is often fused to LIF for expression. TrxA catalyzes the reduction of disulfide bonds in proteins, helping them fold correctly and increasing the solubility of exogenous proteins. Summary of the Invention

[0004] 1. Problem to be solved

[0005] To address the existing problems of hLIF, such as low thermal stability and a short half-life of biological activity, the present application provides a recombinant hLIF mutant. This recombinant hLIF mutant is a point mutation generated on wild-type hLIF using an energy and evolutionary approach. The mutant includes one or more of the following point mutations: threonine at position 65 is mutated to glutamine (T65Q), alanine at position 119 is mutated to leucine (A119L), and serine at position 127 is mutated to isoleucine (S127I). The recombinant hLIF mutant exhibits biological activity, improved thermal stability, and a prolonged biological half-life. Furthermore, the recombinant hLIF mutant is fused with TrxA to produce a biologically active recombinant hLIF mutant with improved thermal stability or a TrxA-hLIF mutant fusion protein, which can be used in cell biology research, medicine, and other fields.

[0006] 2. Technical solution

[0007] In order to solve the above problems, the technical solutions adopted in this application are as follows:

[0008] The present application provides a recombinant hLIF mutant, which includes one or more point mutations of wild-type hLIF, including a mutation of threonine at position 65 to glutamine (T65Q), a mutation of alanine at position 119 to leucine (A119L), and a mutation of serine at position 127 to isoleucine (S127I). The above point mutations are point mutations that occur on wild-type hLIF based on energy and evolution methods, thereby improving the thermal stability of hLIF.

[0009] Furthermore, the amino acid sequence of the wild-type hLIF is shown in SEQ ID NO.1.

[0010] Furthermore, the above-mentioned recombinant hLIF mutant is a wild-type hLIF in which the threonine at position 65 is mutated to glutamine (T65Q), and is named hLIF mutant M1. Its amino acid sequence is shown in SEQ ID NO.2.

[0011] Furthermore, the above-mentioned recombinant hLIF mutant is a wild-type hLIF in which the alanine at position 119 is mutated to leucine (A119L), and is named hLIF mutant M2. Its amino acid sequence is shown in SEQ ID NO.3.

[0012] Furthermore, the above-mentioned recombinant hLIF mutant is a wild-type hLIF in which the serine at position 127 is mutated to isoleucine (S127I), and is named hLIF mutant M3. Its amino acid sequence is shown in SEQ ID NO.4.

[0013] Furthermore, the above-mentioned recombinant hLIF mutant is a wild-type hLIF in which the threonine at position 65 is mutated to glutamine (T65Q), and the alanine at position 119 is mutated to leucine (A119L), named hLIF mutant M4, and its amino acid sequence is shown in SEQ ID NO.5.

[0014] Furthermore, the above-mentioned recombinant hLIF mutant is a wild-type hLIF in which the threonine at position 65 is mutated to glutamine (T65Q), the alanine at position 119 is mutated to leucine (A119L), and the serine at position 127 is mutated to isoleucine (S127I), and is named hLIF mutant M5. Its amino acid sequence is shown in SEQ ID NO.6.

[0015] The present application also provides a fusion protein TrxA-hLIF mutant, comprising TrxA and any of the above-mentioned recombinant hLIF mutants. Compared with the wild-type fusion protein TrxA-hLIF, the fusion protein TrxA-hLIF mutant has an increased Tm value, has biological activity, and has improved thermal stability.

[0016] The present application also provides a nucleic acid molecule encoding the above-mentioned recombinant hLIF mutant. Those skilled in the art know that due to the degeneracy of codons, there may be many nucleotide sequences that can encode the hLIF mutant of the present invention.

[0017] Furthermore, the nucleotide sequence of the DNA molecule encoding the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.7.

[0018] Furthermore, the nucleotide sequence of the DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.8.

[0019] Furthermore, the nucleotide sequence of the DNA molecule encoding the amino acid sequence shown in SEQ ID NO.3 is shown in SEQ ID NO.9.

[0020] Furthermore, the nucleotide sequence of the DNA molecule encoding the amino acid sequence shown in SEQ ID NO.4 is shown in SEQ ID NO.10.

[0021] Furthermore, the nucleotide sequence of the DNA molecule encoding the amino acid sequence shown in SEQ ID NO.5 is shown in SEQ ID NO.11.

[0022] Furthermore, the nucleotide sequence of the DNA molecule encoding the amino acid sequence shown in SEQ ID NO.6 is shown in SEQ ID NO.12.

[0023] The present application also provides a method for preparing the above-mentioned fusion protein TrxA-hLIF wild type or mutant, comprising: connecting the above-mentioned nucleic acid molecule encoding the recombinant hLIF wild type or recombinant hLIF mutant and the nucleic acid molecule encoding TrxA and cloning them into a plasmid, introducing the plasmid into Escherichia coli SHuffle T7 to obtain the Escherichia coli strain SHuffle T7 containing the recombinant plasmid encoding the TrxA-hLIF wild type or TrxA-hLIF mutant, inducing expression using IPTG, and finally extracting and purifying to obtain the fusion protein TrxA-hLIF wild type and mutant. SHuffle T7 is a derivative strain of K12, and a disulfide isomerase DsbC gene is integrated into the chromosome of this strain, which can promote the correct folding of disulfide bond-containing proteins.

[0024] Furthermore, the above-mentioned plasmid includes pET32a and the like.

[0025] Furthermore, the above-mentioned IPTG-induced expression includes: culturing the E. coli strain SHuffle T7 containing the recombinant plasmid encoding the wild-type TrxA-hLIF or the mutant TrxA-hLIF to an OD of 600 At approximately 0.6-0.8, the culture was cooled to 18°C ​​and induced with IPTG at a final concentration of 0.5 mM for 16 h at 180 rpm.

[0026] The present application also provides the use of the above-mentioned fusion protein TrxA-hLIF mutant in cell culture, which is used for culturing embryonic stem cells and can maintain the pluripotency of embryonic stem cells.

[0027] Furthermore, the above-mentioned embryonic stem cells include embryonic stem cells from animals such as mice and pigs.

[0028] 3. Beneficial effects

[0029] Compared with the prior art, the present application has the following advantages:

[0030] The present application provides a recombinant hLIF mutant, which is a point mutation on wild-type hLIF based on energy and evolution methods, including the superposition of one or more point mutations in wild-type hLIF, in which threonine at position 65 is mutated to glutamine (T65Q), alanine at position 119 is mutated to leucine (A119L), and serine at position 127 is mutated to isoleucine (S127I). The recombinant hLIF mutant has biological activity, improved thermal stability, and prolonged biological activity half-life. At the same time, the recombinant hLIF mutant is fused with TrxA to express and prepare a recombinant TrxA-hLIF mutant with improved thermal stability and biological activity, which can be used in cell biology research, medicine and other fields. Experiments have shown that compared with wild-type TrxA-hLIF, the Tm values ​​of the mutants of the several fusion proteins TrxA-hLIF described in the present invention are significantly improved, and the TrxA-hLIF mutants have certain activity in animal cell culture such as mice and pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Shown are the protein expression levels of wild-type TrxA-hLIF and TrxA-hLIF mutants.

[0032] Figure 2 This is a graph analyzing the thermal stability of wild-type TrxA-hLIF and TrxA-hLIF mutants.

[0033] Figure 3 The figure shows how wild-type TrxA-hLIF and TrxA-hLIF mutant M5 promote the proliferation of mES cells (embryonic stem cells) at P6 generation at different concentrations.

[0034] Figure 4 The wild-type TrxA-hLIF and the TrxA-hLIF mutant M5 maintain the expression of key genes for cell pluripotency in the P6 generation. DETAILED DESCRIPTION

[0035] The present application is further described below with reference to specific embodiments.

[0036] It should be noted that the terms such as "upper", "lower", "left", "right", and "middle" cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0038] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0039] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement, or value. One skilled in the art can readily determine the degree of flexibility for a particular variable.

[0040] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" explicitly includes only A, only B, only C, and combinations of each thereof.

[0041] Concentration, amount and other numerical data can be presented in range format in this article.Should be understood that such range format is only used for convenience and brevity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all independent numerical values ​​or subranges encompassed within the scope, just as each numerical value and subrange are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including independent numerals (such as 2,3,4) and subranges (such as 1 to 3,2 to 4 etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5," which should be interpreted as including all above-mentioned values ​​and scopes.In addition, no matter how the breadth of described scope or feature is, this explanation should be applicable.

[0042] Example 1

[0043] This example provides the construction of strains expressing wild-type TrxA-hLIF and TrxA-hLIF mutants, which specifically includes the following steps:

[0044] The amino acid sequence of wild-type hLIF is shown in SEQ ID NO. 1. A nucleic acid molecule encoding the amino acid sequence was designed based on the amino acid sequence, and its nucleotide sequence is shown in SEQ ID NO. 7. The nucleic acid molecule was synthesized by General Biotechnology (Anhui) Co., Ltd. The gene encoding wild-type hLIF was cloned downstream of the TrxA gene in the commercially available pET32a vector via enzyme digestion and ligation. The plasmid was transformed into Escherichia coli SHuffle T7 to obtain a strain expressing wild-type TrxA-hLIF.

[0045] The same method was used to obtain a mutant strain M1 expressing hLIF mutant M1. The hLIF mutant M1 is a wild-type hLIF mutant in which the threonine at position 65 is mutated to glutamine (T65Q). Its amino acid sequence is shown in SEQ ID NO.2, and its encoding nucleotide sequence is shown in SEQ ID NO.8.

[0046] A mutant strain M2 expressing hLIF mutant M2 was obtained. The hLIF mutant M2 is a wild-type hLIF in which the alanine at position 119 is mutated to leucine (A119L). Its amino acid sequence is shown in SEQ ID NO.3, and its encoding nucleotide sequence is shown in SEQ ID NO.9.

[0047] A mutant strain M3 expressing hLIF mutant M3 was obtained. The serine at position 127 of wild-type hLIF was mutated to isoleucine (S127I). Its amino acid sequence is shown in SEQ ID NO.4, and its encoding nucleotide sequence is shown in SEQ ID NO.10.

[0048] A mutant strain M4 expressing the hLIF mutant M4 was obtained. The hLIF mutant M4 is a wild-type hLIF in which the threonine at position 65 is mutated to glutamine (T65Q), and the alanine at position 119 is mutated to leucine (A119L). Its amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence encoding it is shown in SEQ ID NO.11.

[0049] A mutant strain M5 expressing the hLIF mutant M5 was obtained. The hLIF mutant M5 is a wild-type hLIF in which the threonine at position 65 is mutated to glutamine (T65Q), the alanine at position 119 is mutated to leucine (A119L), and the serine at position 127 is mutated to isoleucine (S127I). Its amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence encoding it is shown in SEQ ID NO.12.

[0050] Example 2

[0051] This example provides the preparation and purification of wild-type TrxA-hLIF and its mutant proteins (M1-M5).

[0052] To obtain wild-type TrxA-hLIF and its mutant proteins, E. coli strains expressing wild-type TrxA-hLIF and hLIF mutants M1-M5 obtained in Example 1 were cultured in LB medium supplemented with ampicillin at 37°C until an OD600 of approximately 0.6-0.8 was reached. The cultures were then cooled to 18°C ​​and induced with IPTG at a final concentration of 0.5 mM for 16 hours at 180 rpm. Cells were harvested by centrifugation (10 minutes, 4°C, 4,000 rpm). Cell pellets from 100 mL of cell culture were resuspended in 10 mL of Tris-HCl lysis buffer (50 mM Tris, 200 mM NaCl, pH 8). Cells were disrupted by sonication on ice, and the lysate was centrifuged (10 minutes, 4°C, 12,000 rpm). The supernatant was applied to a His-Accept nickel column (Beyotime, P2233-100 ml). After washing the unbound proteins with 10 mL of lysis buffer, the bound proteins were eluted with elution buffer (50 mM Tris-HCl pH 8, 300 mM NaCl, 200 mM imidazole), and the buffer was exchanged into 1× PBS solution. The wild-type TrxA-hLIF and its mutant proteins were obtained using a PD-10 gel filtration column (GE Healthcare).

[0053] The results are as follows Figure 1 The results showed that wild-type TrxA-hLIF and mutants M1-M5 could be well expressed in a soluble manner using SHuffle T7 as the host and TrxA tag. The size of TrxA-hLIF and its mutants was 34.1 kDa.

[0054] Example 3

[0055] This example provides a thermal stability assay for wild-type TrxA-hLIF and fusion protein TrxA-hLIF mutants M1-M5, specifically including the following methods:

[0056] The protein solution was mixed with Sypro Orange dye (Merck, Cat. No. S5692-500UL) in a 9:1 ratio. The prepared samples were transferred to a 96-well PCR plate for testing, with a sample volume of 20 μL per test well. 1× PBS buffer was used as a blank control group, with three replicates. Each test group also had three replicates. Then, the 96-well PCR plate containing the control and test groups was placed in a real-time fluorescence quantitative PCR instrument. The excitation and emission wavelengths were adjusted to 492 nm and 610 nm, respectively. The heating rate was set to 1°C / min. The temperature range was generally from 25°C to 90°C, and the fluorescence intensity was measured every 1°C increase. Finally, using analysis software such as Origin Pro, a curve of fluorescence intensity versus temperature can be plotted to determine the Tm value of the sample protein.

[0057] The results are as follows Figure 2 It was shown that mutation of the threonine at position 65 of wild-type hLIF to glutamine (T65Q), the alanine at position 119 to leucine (A119L), and the serine at position 127 to isoleucine (S127I) could all increase the Tm value of the fusion protein, and combining the three points to form a triple-point mutant increased its Tm value by 4°C.

[0058] Example 4

[0059] This example provides a biological activity assay for wild-type TrxA-hLIF and TrxA-hLIF mutant M5, specifically including the following methods:

[0060] To evaluate the ability of the thermostable TrxA-hLIF mutant M5 to maintain the undifferentiated state of mouse embryonic stem cells (mES cells), revived mES cells were seeded into 12-well cell culture plates and cultured in a 37°C, 5% CO2 cell culture incubator. DMEM cell culture medium was used to add wild-type TrxA-hLIF and TrxA-hLIF mutant M5 at a final concentration of 0-40 ng / mL. Commercial hLIF (final concentration of 10 ng / mL, purchased from GenScript, Cat. No. Z02681) was used as a positive control. The medium was changed once a day and passaged every two days. The corresponding number of cells (1×10 5 Cell growth curves were generated for each group based on the number of cells before and after passage, and images of each group of cells were taken before passage. To examine the pluripotency status of mES cells in more detail, the expression of pluripotency markers Oct4, Nanog, Klf4, and Sox2 at passages P3 and P6 was measured by qPCR and Western blotting.

[0061] qPCR: Total RNA was extracted from P3 and P6 cells using an RNA extraction kit (Novagen, Cat. No. RC101). After extraction, the RNA concentration of each sample was determined. An appropriate amount of RNA solution was reverse-transcribed into cDNA using a reverse transcription kit (Novagen, Cat. No. R323). After configuring the system using a qPCR kit (Novagen, Cat. No. Q311), the qPCR instrument was set up in "SYBR" mode. The target genes were Oct4, Nanog, Klf4, and Sox2. The internal reference gene was GAPDH.

[0062] Cell lysis: Lyse P3 and P6 mES cells using cell lysis buffer (RIPA lysis buffer: PMSF = 100:1, 100 μL lysis buffer per sample). Place the collected cell tube on ice for 30 min, then centrifuge at 12,000 rpm, 4°C for 10 min, and collect the cell lysate supernatant.

[0063] Protein electrophoresis: The protein concentration in the cell lysate supernatant was determined using a BCA protein concentration assay kit (Thermo Fisher Scientific, Cat. No. 23225). The proteins were adjusted to the same concentration and 5× protein loading buffer was added. The samples were heated at 100°C for 5 min and subjected to 12% SDS-PAGE electrophoresis.

[0064] Transfer: After protein electrophoresis, activate the PVDF membrane in methanol for 30 seconds and then immerse it in the equilibration solution.

[0065] A sponge pad, PVDF membrane, protein gel, and sponge pad were placed on the transfer cassette in that order, with the protein gel placed near the cathode end of the transfer chamber. The membrane was transferred using a transfer apparatus (GenScript) for 12 minutes. After transfer, the PVDF membrane was blocked with 5% skim milk for 1 hour and then washed three times with 1× TBST for 10 minutes each. The corresponding primary antibodies (Oct4, Nanog, Sox2, GAPDH) were added and incubated overnight at 4°C. The membrane was then washed three times with 1× TBST for 10 minutes each. HRP-conjugated secondary antibodies were added and incubated at room temperature for 2 hours. The membrane was then washed three times with 1× TBST for 10 minutes each. The membrane was developed using an ECL luminescence kit (Beyotime, Cat. No. P0018AS), and the results were recorded and analyzed. Western blotting was performed to analyze the expression levels of genes associated with stemness maintenance in mES cells treated with different concentrations of TrxA-hLIF (WT and M5).

[0066] The results are as follows Figure 3 The results showed that the TrxA-hLIF mutant M5 had more proliferative cells and better morphology than the wild-type TrxA-hLIF at the added concentrations of 10, 20, and 40 ng / mL.

[0067] Figure 4The results showed that at P6, when added at low concentrations of 2.5, 5, and 10 ng / mL, the stemness gene expression level of the TrxA-hLIF mutant M5 was significantly higher than that of the wild-type TrxA-hLIF, indicating that the TrxA-hLIF mutant M5 has a longer biological activity half-life and can maintain the stemness of embryonic stem cells at lower concentrations.

Claims

1. A recombinant hLIF mutant, characterized in that: The recombinant hLIF mutant is a wild-type hLIF in which the threonine at position 65 is mutated to glutamine (T65Q), the alanine at position 119 is mutated to leucine (A119L), and the serine at position 127 is mutated to isoleucine (S127I). The amino acid sequence of the wild-type hLIF is shown in SEQ ID NO.1, and the amino acid sequence of the recombinant hLIF mutant is shown in SEQ ID NO.

6.

2. A fusion protein TrxA-hLIF mutant, characterized in that it comprises TrxA and the recombinant hLIF mutant according to claim 1.

3. A nucleic acid molecule, characterized in that The nucleotide sequence of the nucleic acid encoding the recombinant hLIF mutant according to claim 1 is shown in SEQ ID NO.

12.

4. The method for preparing a fusion protein TrxA-hLIF mutant according to claim 2, characterized in that: The method comprises the following steps: connecting the nucleic acid molecule encoding the recombinant hLIF mutant according to claim 3 and the nucleic acid molecule encoding TrxA and cloning them into a plasmid, introducing the plasmid into Escherichia coli SHuffle T7 to obtain the Escherichia coli strain SHuffle T7 containing the recombinant plasmid encoding the TrxA-hLIF mutant, inducing expression using IPTG, and finally extracting and purifying to obtain the fusion protein TrxA-hLIF mutant.

5. Use of the fusion protein TrxA-hLIF mutant according to claim 2 in embryonic stem cell culture.

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