Method for expressing complex of human IGF-1 and IGF-1 binding segment of human igfbp3 in pichia pastoris

Abstract

Provided is a method for expressing a complex of human IGF-1 and an IGF-1 binding segment of human IGFBP3 in Pichia pastoris. In order to improve the recombinant expression capacity of human IGF-1, the protein complex of IGF-1 and an IGF-1 binding segment of an endogenous binding protein IGFBP3 of IGF-1 is obtained in Pichia pastoris by means of the methods of fusion expression and co-expression of a target protein with the endogenous binding protein. The complexes obtained by means of the two expression methods in Pichia pastoris both exhibit cell proliferation activity, with the expression levels both reaching 1 g / L. The obtained protein complex of IGF-1 and the IGF-1 binding segment of IGFBP3 has thermostability, acid resistance, and alkali resistance.

Description

A method for expressing a complex of human IGF-1 and the IGF-1 binding region of IGFBP3 in Pichia pastoris. Technical Field

[0001] This invention relates to the field of genetic engineering, and more specifically to a method for expressing a complex of human IGF-1 and the IGF-1 binding segment of IGFBP3 in Pichia pastoris. Background Technology

[0002] IGF-1 (insulin-like growth factor-1), also known as somatostatin C, is a type of insulin-like growth factor with a chemical structure similar to insulin and is one of the important cytokines in the body. Human IGF-1 is a basic polypeptide chain composed of 70 amino acids, with 6 cysteine ​​residues (Cys) forming three pairs of intrachain disulfide bonds. Most of the IGF-1 in serum is bound to the protein IGFBP3 (IGF binding protein 3) and ALS (acid-labile subunit) to form the IGF-1 / IGFBP3 / ALS ternary complex. This complex can prolong the serum half-life of IGF-1 and regulate its function. Under specific conditions, IGFBP3 in the ternary complex is cleaved by a specific protease, thereby releasing IGF-1 from the complex. The released IGF-1 specifically binds to the IGF-1 receptor IGF1R on the cell surface, altering the conformation of the IGF1R β subunit, activating tyrosine kinase activity, and cascading activation of the PI3K-MAPK pathway, thereby exerting its various biological activities. LR3 IGF-1 (Long R3IGF-1) is an analogue of IGF-1.

[0003] To enhance the recombinant expression of human IGF-1 or LR3 IGF-1, existing techniques have reported methods to promote the expression of target proteins in Pichia pastoris through fusion proteins. This involves fusing the xylanase gene XynCDBFV to the N-terminus of IGF-1 or LR3 IGF-1 to obtain XynCDBFV-IGF-1 or LR3 IGF-1 proteins. In this technique, when xylanase is fused with IGF-1, IGF-1 expression is not detected when the fusion is linked using the endogenous protease KEX2 site. Only by using the exogenous protease TEV site can the expression of the xylanase-IGF-1 fusion protein be obtained. After purification, the fusion protein is digested with a commercially available TEV protease to obtain individual IGF-1. The expression level of the xylanase-IGF-1 fusion protein is 0.5 g / L, requiring further digestion with exogenous TEV protease and purification to obtain active IGF-1. Summary of the Invention

[0004] The purpose of this invention is to provide a method for expressing the IGF-1 binding segment complex of human IGF-1 and IGFBP3 in Pichia pastoris.

[0005] The method for expressing the IGF-1 binding segment complex of human IGF-1 and IGFBP3 in Pichia pastoris according to the present invention comprises the following steps:

[0006] The optimized Pichia pastoris-preferred gene encoding human IGF-1 and the optimized Pichia pastoris-preferred gene encoding the IGF-1 binding region of human IGFBP3 are synthesized, wherein the amino acid sequence of the human IGF-1 is shown in SEQ ID NO: 1 and the amino acid sequence of the IGF-1 binding region of the human IGFBP3 is shown in SEQ ID NO: 2.

[0007] Construct fusion expression or co-expression vectors, wherein the fusion expression vector includes an expression cassette expressing the IGF-1 binding region of human IGF-1 and human IGFBP3, and the co-expression vector includes an expression cassette of human IGF-1 and an expression cassette of human IGFBP3.

[0008] The constructed fusion expression or co-expression vectors were linearized and then transformed into Pichia pastoris to obtain recombinant Pichia pastoris. The recombinant Pichia pastoris was cultured to express the IGF-1 binding segment complex of human IGF-1 and IGFBP3.

[0009] Human IGF-1, SEQ ID NO: 1:

[0010] The IGF-1 binding region of human IGFBP3, shown in SEQ ID NO: 2:

[0011] According to the present invention, a method for expressing a complex of human IGF-1 and IGFBP3 IGF-1 binding segment in Pichia pastoris, wherein the nucleotide sequence of the optimized Pichia pastoris-preferred gene encoding human IGF-1 is shown in SEQ ID NO: 3; and the nucleotide sequence of the optimized Pichia pastoris-preferred gene encoding the IGF-1 binding segment of human IGFBP3 is shown in SEQ ID NO: 4.

[0012] Human IGF-1 gene, SEQ ID NO: 3:

[0013] Human IGFBP3 IGF-1 binding segment gene, SEQ ID NO: 4:

[0014] According to the present invention, a method for expressing a complex of human IGF-1 and IGFBP3 IGF-1 binding segment in Pichia pastoris, wherein the expression cassette of the fusion expression vector comprises the following operatively linked elements: an AOX1 promoter, a Saccharomyces cerevisiae α-Factor signal peptide encoding gene, a gene encoding the human IGFBP3 IGF-1 binding segment preferred by the optimized Pichia pastoris, a gene encoding human IGF-1 preferred by the optimized Pichia pastoris, and a terminator.

[0015] According to the present invention, a method for expressing a complex of human IGF-1 and IGFBP3 IGF-1 binding segment in Pichia pastoris, wherein the expression cassette of the human IGF-1 in the co-expression vector comprises the following operatively linked elements: an AOX1 promoter, a *Saccharomyces cerevisiae* α-Factor signal peptide encoding gene, the optimized *Pichia pastoris*-preferred gene encoding human IGF-1, and a terminator; the expression cassette of the human IGFBP3 IGF-1 binding segment in the co-expression vector comprises the following operatively linked elements: an AOX1 promoter, a *Saccharomyces cerevisiae* α-Factor signal peptide encoding gene, the optimized *Pichia pastoris*-preferred gene encoding the human IGFBP3 IGF-1 binding segment, and a terminator.

[0016] The present invention relates to a method for expressing a complex of human IGF-1 and IGFBP3 IGF-1 binding segment in Pichia pastoris. The expression cassettes for both expression methods contain an endogenous protease KEX2 site (KREAEA) within the gene linker between the Saccharomyces cerevisiae α-Factor signal peptide encoding gene and the human IGF-1 / human IGFBP3 IGF-1 binding segment gene.

[0017] The fusion expression cassette contains a linker sequence (RSG) and an endogenous protease KEX2 site (KREAEA) between the IGFBP3 gene and the human IGF-1 gene sequence.

[0018] The terminator of fusion expression contains a His tag sequence, the terminator of co-expressed human IGF-1 contains a His tag sequence, and the terminator of the IGF-1 binding region of human IGFBP3 does not contain a His tag sequence.

[0019] According to the technical solution of this application, the term "IGF-1 binding segment of IGFBP3" refers to the N-terminus of IGFBP3 that binds to IGF-1, not the entire length of IGFBP3.

[0020] According to a specific embodiment of this application, full-length human IGFBP3 was fused with human IGF-1 and its analogue LR3IGF-1, respectively, with the fusion linker connected to the endogenous protease KEX2 site. The result was a fusion protein of IGFBP3 and IGF-1. However, because the C-terminus of IGFBP3 may be cleaved, the fusion protein was a complex of the N-terminus of IGFBP3 and IGF-1, rather than a complex of full-length IGFBP3 and IGF-1. The fusion protein of IGFBP3 and LR3IGF-1 was not detected. Therefore, this application determines that the IGF-1 binding region of IGFBP3 is used for fusion expression with IGF-1.

[0021] According to the specific embodiments of this application, co-expression of IGF-1 with the IGF-1 binding region of IGFBP3 also yields a complex of the N-terminus of IGFBP3 and IGF-1. Unexpectedly, when attempting to purify IGF-1 using His-tag affinity chromatography at the C-terminus of IGF-1, a single IGF-1 protein was not obtained; instead, a complex of the IGF-1 binding region of IGFBP3 and IGF-1 was obtained, and it was not completely separated on an SDS-PAGE gel.

[0022] The beneficial technical effects of this application are as follows:

[0023] According to the technical solution of this application, the IGF-1 binding region of IGFBP3 is fused with IGF-1, and IGF-1 expression can still be detected even if the intermediate link is connected by the endogenous protease KEX2 site. Furthermore, using a co-expression method, the N-terminus of IGFBP3 and IGF-1 are simultaneously expressed in the Pichia pastoris system, and IGF-1 expression can be detected in vitro. Proteins obtained from both expression methods are subjected to affinity chromatography via a His tag linked to the C-terminus of IGF-1 to directly obtain the complex protein of the IGF-1 binding region of IGFBP3 and IGF-1 bound together.

[0024] This invention designs two pathways for expressing the N-terminus of human IGFBP3 and the human IGF-1 complex in Pichia pastoris: fusion expression and co-expression. Fusion expression achieves protein cleavage by adding a yeast endogenous protease cleavage site (KREAEA). Co-expression involves designing two expression cassettes in the expression vector to simultaneously express the IGF-1 binding region of human IGFBP3 (the N-terminus of IGFBP3) and the human IGF-1 protein. Both methods result in a tight binding between the expressed IGFBP3 IGF-1 binding region and IGF-1. Affinity chromatography using a His tag attached to the C-terminus of IGF-1 directly yields the bioactive IGFBP3 IGF-1 binding region and IGF-1 complex protein, which exhibits strong temperature, acid, and alkali resistance.

[0025] Furthermore, the expression level of the IGF-1 binding region of IGFBP3 and the IGF-1 complex reached 1 g / L, requiring only one purification step and eliminating the need for commercial enzyme digestion, thus improving yield and reducing cost. Attached Figure Description

[0026] Figure 1 is an SDS-PAGE image of human IGFBP3 fusion expression with IGF-1 and LR3 IGF-1, where A is IGF-1 and B is LR3 IGF-1.

[0027] Figure 2 shows the SDS-PAGE of the IGF-1 binding region and the IGF-1 complex of purified human IGFBP3, where A represents the 1× and 2× concentrations of the purified protein after fusion expression, and B represents the 1× and 2× concentrations of the purified protein after co-expression.

[0028] Figure 3 shows the mass spectrometry identification of the IGF-1 binding region of purified human IGFBP3 and the complex with IGF-1. The shaded amino acids are the amino acid peptides detected by mass spectrometry. Band a, Band b, and Band c are the protein bands after fusion expression and purification as shown in Figure 2, and Band d and Band e are the protein bands after co-expression and purification.

[0029] Figure 4 shows the cell proliferation activity of the IGF-1 binding region of purified human IGFBP3 and the complex with IGF-1. A represents the fusion expressed protein, and B represents the co-expressed protein.

[0030] Figure 5 shows the DSC curve of the IGF-1 binding region of purified human IGFBP3 and the IGF-1 complex, where A is the fusion expressed protein and B is the co-expressed protein.

[0031] Figure 6 shows the effect of the IGF-1 binding region of purified human IGFBP3 and the IGF-1 complex on cell proliferation activity after treatment at different temperatures (4℃, 25℃, 37℃, 60℃) for 1, 3, and 7 days.

[0032] Figure 7 shows the effect of the IGF-1 binding region of purified human IGFBP3 and the IGF-1 complex on cell proliferation activity under acidic (pH=2, 3, 4) and alkaline (pH=9, 10, 11) conditions. Detailed Implementation

[0033] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0034] Unless otherwise specified, the biological materials, reagents, or devices used in the following examples are all available from conventional commercial sources or can be obtained by existing known methods. Molecular biology experimental methods not specifically described in the following examples were performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions.

[0035] In the following examples, the optimized nucleotide sequence of the gene encoding human IGF-1 is shown in SEQ ID NO: 3, the optimized nucleotide sequence of the gene encoding the IGF-1 binding region of human IGFBP3 is shown in SEQ ID NO: 4, and the optimized nucleotide sequence of the LR3 IGF-1 gene is shown in SEQ ID NO: 5. All optimized nucleotide sequences were obtained through chemical synthesis, and then the DNA sequences were introduced into expression vectors and cells.

[0036] SEQ ID NO: 5, Optimized LR3 IGF-1 gene nucleotide sequence:

[0037] Example 1: Construction of a fusion expression vector

[0038] The expression cassette containing the IGF-1 binding region of human IGFBP3 and the expression cassette of human IGF-1 gene includes the following operationally linked elements: AOX1 promoter, Saccharomyces cerevisiae α-Factor signal peptide encoding gene, IGF-1 binding region gene of human IGFBP3, IGF-1 gene, and terminator.

[0039] The Saccharomyces cerevisiae α-Factor signal peptide encoding gene contains an endogenous protease cleavage site (KREAEA) between the IGFBP3 IGF-1 binding region gene and the IGF-1 gene sequence; a linker sequence (RSG) and an endogenous protease site (KREAEA) are contained between the IGFBP3 IGF-1 binding region gene and the IGF-1 gene sequence; and a His tag sequence is contained before the terminator.

[0040] Following the above construction strategy, the desired element was constructed between the Pichia pastoris expression vector pPIC9KEcoRI and SpeI using homologous recombination to obtain a recombinant plasmid. This plasmid was then transformed into E. coli Top10 strain to obtain a recombinant E. coli strain, from which the recombinant plasmid was extracted.

[0041] Example 2: Construction of co-expression vector

[0042] The expression vector contains an expression cassette containing the gene encoding the IGF-1 binding region of human IGFBP3 and an expression cassette containing the human IGF-1 gene. The expression cassette containing the human IGF-1 gene includes the following operatively linked elements: an AOX1 promoter, a *Saccharomyces cerevisiae* α-Factor signal peptide encoding gene, a human IGF-1 gene, and a terminator. The expression cassette containing the gene encoding the IGF-1 binding region of human IGFBP3 includes the following operatively linked elements: an AOX1 promoter, a *Saccharomyces cerevisiae* α-Factor signal peptide encoding gene, a human IGFBP3 IGF-1 binding region gene, and a terminator.

[0043] Both expression cassettes contain an endogenous protease cleavage site (KREAEA) between the gene encoding the α-Factor signal peptide in *Saccharomyces cerevisiae* and the IGF-1 binding region of IGFBP3 or the IGF-1 gene; and contain a His tag sequence before the expression of the IGF-1 terminator.

[0044] Following the aforementioned construction strategy, a gene expression cassette element containing the human IGF-1 gene was constructed between the Pichia pastoris expression vectors pPIC9KEcoRI and SpeI using homologous recombination to obtain a human IGF-1 recombinant plasmid. Then, a gene expression cassette element containing the IGF-1 binding region of the human IGFBP3 gene was constructed between the BamHI-digested IGF-1 recombinant plasmid using homologous recombination to obtain a recombinant plasmid containing two expression cassettes. This plasmid was transformed into E. coli Top10 strain to obtain a recombinant E. coli strain, from which the recombinant plasmid was extracted.

[0045] The recombinant plasmids constructed by the above-mentioned fusion expression and co-expression vectors are operated in the same way in the following implementations.

[0046] Example 3: Expression and isolation of recombinant proteins

[0047] The recombinant plasmid obtained above was linearized by digestion with Bgl II restriction enzyme. The linearized recombinant plasmid DNA fragment was then transformed into fresh Pichia pastoris GS115 competent cells using electroporation. After approximately 2–3 days, clones grew on MD plates. Clones were then picked for induction expression detection. The MD culture plate containing clones was removed from the incubator, and clones were picked using sterile toothpicks. The toothpicks were then placed into yeast culture tubes, each containing 3 mL of BMGY medium. The cells were incubated at 30°C and 220 rpm for 48 hours. The culture was then centrifuged at 4,500 rpm for 5 minutes, the supernatant was discarded, and 1 mL of fresh BMGY induction medium was added to the yeast culture tubes. The cells were incubated at 30°C and 220 rpm for another 48 hours. After 48 hours of induction, the yeast culture was transferred to a 2 mL centrifuge tube and centrifuged at 12,000 rpm for 2 minutes. The fermentation supernatant was then used for SDS-PAGE analysis of proteins.

[0048] After obtaining positive clones through tube-based induction expression, the positive clones were subjected to shake-flask expression detection. The expression strain was inoculated into fresh YPD liquid medium (30 mL) for activation culture at 30°C and 220 rpm for 48 hours. The activated bacterial culture was then transferred to BMGY liquid medium (400 mL) at a 1% inoculation rate and cultured at 30°C and 220 rpm for 48 hours. The bacterial cells were then collected, centrifuged at 4,500 rpm and 4°C for 5 minutes, the supernatant was discarded, and BMMY medium (pre-added with 1% methanol) was added. The culture was then induced and shake-flask cultured at 30°C and 220 rpm for 48–72 hours, with methanol added every 12 hours at a rate of 0.5%. The induction culture was centrifuged at 12,000 rpm and 4°C for 10 minutes, and the culture supernatant was collected for SDS-PAGE protein electrophoresis analysis and subsequent protein purification.

[0049] After shake-flask fermentation, human IGF-1 was isolated and purified from the fermentation medium. Since the IGF-1 binding region of the fusion protein IGFBP3 and the target protein human IGF-1 had already been separated from each other in the shake-flask expression supernatant by enzymatic cleavage with the endogenous protease Kex2, and a 6xHis tag was designed for the C-terminus of human IGF-1, the fermentation supernatant was directly filtered through a 0.22 μM filter and purified using a Ni column. The purification steps were as follows: 15 mL of binding buffer (20 mM Tris HCl, 0.5 M NaCl, 10 mM imidazole, pH 8.0) was added to wash the Ni column; the filtered supernatant was added and passed through the column; the sample was retained (breakthrough).

[0050] Add 10 mL of binding buffer (20 mM Tris HCl, 0.5 M NaCl, 20 mM imidazole, pH 8.0) and wash the sample through the column; retain the column buffer (bound). Add 5 mL of wash buffer (20 mM Tris HCl, 0.5 M NaCl, 40 mM imidazole, pH 8.0) and wash the sample through the column; retain the column buffer (rinse). Add 6 mL of elution buffer (20 mM Tris HCl, 0.5 M NaCl, 300 mM imidazole, pH 8.0) and elute the target protein in 3 tubes. Perform SDS-PAGE electrophoresis on the samples. Determine the sample to be retained based on the electrophoresis results. Mix the samples and add the pre-packed Ni-NTA agarose solution with 20 mM Tris HCl, 0.5 M NaCl, 10 mM imidazole, pH 8.0. 8.0 Equilibrate with binding buffer; Filter the fermentation supernatant through a 0.22 μM filter membrane and pass it through Ni-NTAA garose; Wash away unbound proteins on the surface of the garose with binding buffer containing 10 mM imidazole and wash away impurities with rinsing buffer containing 40 mM imidazole, then wash out the target protein with elution buffer containing 300 mM imidazole, and finally dialyze to PBS buffer for storage.

[0051] As shown in Figure 1, human full-length IGFBP3 and IGF-1 were detected to be expressed. However, based on protein size analysis, the full-length IGFBP3 protein was fragmented, possibly cleaved at the intermediate protease-sensitive site. Human full-length IGFBP3 and LR3 IGF-1 were not detected to be expressed.

[0052] Example 4: Identification of Recombinant Protein Profiles

[0053] After expressing, purifying, and dialyzing the recombinant protein using the above method, the obtained protein needs to be identified by mass spectrometry. Protein samples preserved in PBS buffer after dialysis were analyzed by SDS-PAGE electrophoresis. The protein bands shown in the electrophoresis image (Figure 2) were identified by gel mass spectrometry. As shown in Figure 2, after purification with Ni-NTA agarose, the SDS-PAGE electrophoresis image revealed a main band at approximately 10 kDa for both fusion expression and co-expression. Mass spectrometry identification showed that all bands were a mixture of the IGF-1 binding region of IGFBP3 and IGF-1.

[0054] The obtained mass spectrometry results were subjected to sequence labeling and relative quantitative analysis to determine the amino acid sequence and relative abundance of the peptide. The amino acid sequence of the human IGF-1 was determined by amino acid sequence alignment as shown in SEQ ID NO: 1, and the amino acid sequence of the IGF-1 binding segment of the human IGFBP3 was shown in SEQ ID NO: 2.

[0055] Figure 3 shows that the main band of purified human IGFBP3 containing both the IGF-1 binding region and IGF-1 protein contains both the IGF-1 binding region and IGF-1 protein, and the amounts are roughly equal. The other bands of fusion expression and co-expression also contain both the IGF-1 binding region and IGF-1 protein of IGFBP3, but the IGF-1 binding region of IGFBP3 contains more protein.

[0056] Since this invention utilizes the His tag at the C-terminus of IGF-1 for purification, in fusion expression, the IGF-1 binding region of IGFBP3 and IGF-1 are cleaved by the endogenous KEX2 protease. In co-expression, the IGF-1 binding region of IGFBP3 and IGF-1 are expressed separately. However, the results showed that the purified protein was a mixture of the IGF-1 binding region of IGFBP3 and IGF-1, and it was not separated in the SDS-PAGE image. This indicates that the IGF-1 binding region of the expressed IGFBP3 is tightly bound to IGF-1, forming a complex.

[0057] Example 5: Assay of Recombinant Protein Cell Proliferation Activity

[0058] Cell proliferation activity assays were performed using the IGF-1 binding fragment of the purified IGFBP3 and its complex with IGF-1, with the mouse embryonic fibroblast cell line NIH 3T3 as the experimental cell line. 1.2 × 10⁻⁶ cells were added to each well of a 96-well plate. 4 Cells were pre-coated in 5 wells for each treatment and cultured for 16 h. The culture medium was then aspirated, washed once with 100 μL PBS, and serum-free DMEM medium was added to each well to dilute the sample. The samples were cultured for 24 h. Dilutes were performed at the following concentration gradients: 0, 5, 12.5, 25, and 50 ng / mL, and cultured for 24 h. The supernatant was aspirated, replaced with DMEM containing cck-8, and a cell-free control was added. After culturing for 1-2 h, the absorbance was measured at 450 nm.

[0059] Figure 4 shows that the mixture of purified IGFBP3 IGF-1 binding region and IGF-1 obtained by fusion expression and co-expression both had a significant proliferative effect on NIH 3T3, and the activity was basically the same as that of commercial IGF-1 protein from R&D Systems, proving that the mixture has biological activity.

[0060] Example 6: Thermal stability analysis of recombinant protein

[0061] Differential scanning calorimetry (DSC) was used to determine the IGF-1 binding region and IGF-1 complex of the purified IGFBP3. Samples were sequentially aspirated into 96-well plates, with 400 μL of solution in each well and a protein concentration of 0.5–2 mg / mL. The purified recombinant protein IGFBP3 IGF-1 binding region and IGF-1 complex were used as the sample, and PBS buffer as the reference solution. The relationship between heat capacity change and temperature was obtained to analyze the thermal stability of the recombinant protein.

[0062] Figure 5 shows that the DSC curves of the mixture of purified human IGFBP3 IGF-1 binding region and IGF-1 protein obtained from fusion expression and co-expression exhibit the same trend, with the temperature at the peak of the curve representing the Tm value of the protein. The peak temperatures of the forward scan curves for both fusion and co-expression proteins are around 74℃, while the peak temperatures of the re-scan curves are around 72℃. The overall peak value of the re-scan curves is lower than that of the forward scan. This indicates that the Tm value of the mixture of human IGFBP3 IGF-1 binding region and IGF-1 protein obtained from fusion and co-expression is approximately 74℃, with similar thermal stability, and both are relatively stable.

[0063] Example 7: Analysis of the effect of temperature on recombinant protein activity

[0064] The IGF-1 binding fragment of the purified IGFBP3 and its complex with IGF-1 were used in experiments. Samples were aliquoted into centrifuge tubes (50 μL per tube) and treated at 4℃, 25℃, 37℃, and 60℃ for 1, 3, and 7 days, respectively. Cell proliferation activity was then compared between treated and untreated samples. The mouse embryonic fibroblast cell line NIH 3T3 was selected as the experimental cell line. 1.2 × 10⁻⁶ cells were added to each well of a 96-well plate. 4 Cells were pre-coated in 5 wells for each treatment and cultured for 16 h. The culture medium was then aspirated, washed once with 100 μL PBS, and then diluted in serum-free DMEM medium in each well. The samples were cultured for 24 h. The following concentration gradients were used: 0, 20, and 40 ng / mL, and cultured for 24 h. The supernatant was aspirated, replaced with DMEM containing cck-8, and a cell-free control was added. After culturing for 1-2 h, the absorbance was measured at 450 nm.

[0065] Figure 6 shows that the mixture of the purified human IGFBP3 IGF-1 binding region and IGF-1 protein expressed in this invention had no significant effect on cell proliferation activity after treatment at 4℃, 25℃, and 37℃ for 1, 3, and 7 days. However, at 60℃, the cell proliferation activity of the protein decreased with prolonged treatment time, but the mixture still maintained approximately 70% of its activity after 7 days of treatment at 60℃. This indicates that the mixture of the human IGFBP3 IGF-1 binding region and IGF-1 protein expressed in this invention exhibits good thermostability and maintains high activity even at higher temperatures.

[0066] Example 8: Analysis of the effect of pH on recombinant protein activity

[0067] Experiments were performed using a mixture of the purified IGFBP3 IGF-1 binding fragment and IGF-1. First, acid-base buffers were prepared. The acidic buffer was disodium hydrogen phosphate-citric acid buffer, prepared in proportions to obtain buffers with pH values ​​of 2, 3, and 4, which were then filtered and stored for later use. The alkaline buffer was glycine-sodium hydroxide buffer, prepared in proportions to obtain buffers with pH values ​​of 9, 10, and 11, which were then filtered and stored for later use. Samples were diluted at a volume ratio of 1:10 (i.e., 10 μL of protein to 90 μL of buffer). The protein was added to the corresponding pH buffer solution and incubated at 37°C for 1 h. Untreated protein was diluted 10-fold with PBS. The mouse embryonic fibroblast cell line NIH 3T3 was selected as the experimental cell line. 1.2 × 10⁻⁶ g of protein was added to each well of a 96-well plate. 4 Cells were pre-coated in 5 wells for each treatment and cultured for 16 h. The culture medium was then aspirated, washed once with 100 μL PBS, and then diluted in serum-free DMEM medium in each well. The samples were cultured for 24 h. Dilutes were performed at the following concentration gradients: 0, 20, and 40 ng / mL, with the control group being the corresponding pH buffer diluted at the same factor. The samples were cultured for 24 h. The supernatant was aspirated, replaced with DMEM containing cck-8, and a cell-free blank control was added. After culturing for 1-2 h, the absorbance was measured at 450 nm.

[0068] Figure 7 shows that the mixture of the purified human IGFBP3 IGF-1 binding region and IGF-1 protein expressed in this invention has no significant effect on cell proliferation activity under strong acid and strong alkali conditions. This indicates that the mixture of the human IGFBP3 IGF-1 binding region and IGF-1 protein expressed in this invention has good acid and alkali resistance.

[0069] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for expressing a complex of human IGF-1 and the IGF-1 binding region of IGFBP3 in Pichia pastoris, characterized in that, The method includes the following steps: The optimized Pichia pastoris-preferred gene encoding human IGF-1 and the optimized Pichia pastoris-preferred gene encoding the IGF-1 binding region of human IGFBP3 are synthesized, wherein the amino acid sequence of the human IGF-1 is shown in SEQ ID NO: 1 and the amino acid sequence of the IGF-1 binding region of the human IGFBP3 is shown in SEQ ID NO:

2. Construct a fusion expression vector, wherein the fusion expression vector contains a fusion expression cassette, the cassette comprising expressing the optimized Pichia pastoris-preferred gene encoding human IGF-1 and the optimized Pichia pastoris-preferred IGF-1 binding region encoding human IGFBP3, or... A co-expression vector was constructed, comprising a co-expression cassette IGF-1 and a co-expression cassette IGFBP3-IGF-1, wherein the co-expression cassette IGF-1 comprises the gene encoding human IGF-1 preferred by the optimized Pichia pastoris, and the co-expression cassette IGFBP3-IGF-1 comprises the gene encoding the IGF-1 binding segment of human IGFBP3 preferred by the optimized Pichia pastoris; The constructed fusion expression or co-expression vectors were linearized and then transformed into Pichia pastoris to obtain two recombinant Pichia pastoris. The recombinant Pichia pastoris were cultured to obtain the complex of human IGF-1 and the IGF-1 binding segment of IGFBP3.

2. The method for expressing a complex of human IGF-1 and the IGF-1 binding region of IGFBP3 in Pichia pastoris according to claim 1, characterized in that, The nucleotide sequence of the optimized Pichia pastoris-preferred gene encoding human IGF-1 is shown in SEQ ID NO: 3; the nucleotide sequence of the optimized Pichia pastoris-preferred gene encoding the IGF-1 binding region of human IGFBP3 is shown in SEQ ID NO:

4.

3. The method for expressing a complex of human IGF-1 and the IGF-1 binding region of IGFBP3 in Pichia pastoris according to claim 1, characterized in that, The fusion expression cassette includes the following elements connected from the 5' end to the 3' end: an AOX1 promoter, a Saccharomyces cerevisiae α-Factor signal peptide encoding gene, a gene encoding the IGF-1 binding segment of human IGFBP3 preferred by the optimized Pichia pastoris, a gene encoding human IGF-1 preferred by the optimized Pichia pastoris, and a terminator.

4. The method for expressing a complex of human IGF-1 and the IGF-1 binding region of IGFBP3 in Pichia pastoris according to claim 1, characterized in that, The co-expression cassette IGF-1 includes the following elements linked from the 5' end to the 3' end: an AOX1 promoter, a *Saccharomyces cerevisiae* α-Factor signal peptide encoding gene, the optimized *Pichia pastoris*-preferred gene encoding human IGF-1, and a terminator; the co-expression cassette IGFBP3-IGF-1 includes the following elements linked from the 5' end to the 3' end: an AOX1 promoter, a *Saccharomyces cerevisiae* α-Factor signal peptide encoding gene, the optimized *Pichia pastoris*-preferred gene encoding the IGF-1 binding region of human IGFBP3, and a terminator.

5. The method for expressing a complex of human IGF-1 and the IGF-1 binding region of IGFBP3 in Pichia pastoris according to claim 3, characterized in that, The gene encoding the α-Factor signal peptide in *Saccharomyces cerevisiae* contains a protease cleavage site, KREAEA, between it and the gene encoding the IGF-1 binding region of human IGFBP3, which is preferred by *Pichia pastoris*.

6. The method for fusing a complex expressing the IGF-1 binding region of human IGF-1 and IGFBP3 in Pichia pastoris according to claim 3, characterized in that, The optimized Pichia pastoris-preferred gene encoding the IGF-1 binding region of human IGFBP3 contains the linker sequence RSG and the protease site KREAEA between the optimized Pichia pastoris-preferred gene encoding human IGF-1.

7. The method for expressing a complex of human IGF-1 and the IGF-1 binding region of IGFBP3 in Pichia pastoris according to claim 4, characterized in that, For the co-expression cassette IGF-1, a protease cleavage site KREAEA is included between the *Saccharomyces cerevisiae* α-Factor signal peptide encoding gene and the optimized *Pichia pastoris* preferred gene encoding human IGF-1; for the co-expression cassette IGFBP3-IGF-1, a protease cleavage site KREAEA is included between the *Saccharomyces cerevisiae* α-Factor signal peptide encoding gene and the optimized *Pichia pastoris* preferred gene encoding the IGF-1 binding region of human IGFBP3.

8. The method for expressing a complex of human IGF-1 and the IGF-1 binding region of IGFBP3 in Pichia pastoris according to claim 3, characterized in that, A His tag sequence is present between the optimized Pichia pastoris-preferred gene encoding human IGF-1 and the terminator.

9. The method for expressing a complex of human IGF-1 and the IGF-1 binding region of IGFBP3 in Pichia pastoris according to claim 4, characterized in that, In the co-expression cassette IGF-1, a His tag sequence is contained between the optimized Pichia pastoris-preferred gene encoding human IGF-1 and the terminator.

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