Preparation method of low-O-glycosylation recombinant III-type humanized collagen

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a preparation method of low-O-glycosylation recombinant III-type humanized collagen. The method comprises the following steps: obtaining recombinant plasmids for expressing recombinant III-type humanized collagen, obtaining a strain for overexpressing phosphomannose isomerase (PMI), then carrying out linear treatment on the recombinant plasmids, transferring the recombinant plasmids into the strain, carrying out fermentation culture, and precipitating the recombinant III-type humanized collagen after the fermentation is ended, so as to obtain the recombinant III-type humanized collagen. The recombinant III type humanized collagen with low O-glycosylation is obtained. According to the method, in the preparation process, the metabolic rate of 6-phosphomannose in cells is increased, accumulation of a large amount of 6-phosphomannose in the cells is restrained, O-glycosylation modification of III-type humanized collagen is reduced, the influence of unnecessary glycosylation modification on the protein space structure and biological functions is avoided, and through the method, the yield of the collagen is increased. The expression level of the recombinant III-type humanized collagen can be obviously improved, and the protein yield is improved.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering technology, and specifically relates to a method for preparing low-O-glycosylated recombinant type III humanized collagen. Background Art

[0002] Type III collagen is an important structural protein that is widely present in elastic tissues such as human skin, tendons and blood vessels. It plays a vital role in maintaining skin elasticity, promoting wound healing and maintaining vascular integrity.

[0003] While Pichia pastoris offers advantages such as low culture costs and high expression efficiency as a host for expressing foreign proteins, glycosylation is unavoidable during the process of secretory protein modification. Protein glycosylation in the Pichia pastoris expression system includes both N-glycosylation and O-glycosylation. O-glycosylation, a form of hypermannosylation, is highly immunogenic. Consequently, the modified glycoproteins are easily recognized and bound by mannose receptors in the human body, leading to their clearance by the immune system and triggering an immune response.

[0004] Currently, the main method to reduce O-glycosylation modification in the Pichia pastoris expression system is to knock out PMT1, PMT2 and PMT4, which are enzymes involved in O-mannose synthesis and play a key role. Although this operation will reduce O-glycosylation modification in the Pichia pastoris expression system, it was found in the subsequent culture process that this method is prone to cause cell death, indicating that the method of knocking out PMT1, PMT2 and PMT4 to reduce O-glycosylation modification in the Pichia pastoris expression system is not applicable.

[0005] Therefore, there is an urgent need to find a method to effectively reduce the occurrence of O-glycosylation modification in the Pichia pastoris expression system. Summary of the Invention

[0006] In order to overcome the above technical problems, the present application proposes a method for reducing O-glycosylation modification in the Pichia pastoris expression system by overexpressing phosphomannose isomerase (PMI), and then using this method to prepare recombinant humanized type III collagen with a lower mannose glycopeptide content, which is safer and more effective and has a triple helical structure.

[0007] In a first aspect, the present application provides a method for preparing low O-glycosylated recombinant humanized type III collagen, the preparation method comprising the following steps: Step 1, obtaining a recombinant plasmid expressing recombinant humanized type III collagen; Step 2, obtaining a strain overexpressing phosphomannose isomerase (PMI); Step 3: linearize the recombinant plasmid described in step 1 and transfer it into the strain described in step 2, ferment and culture it, and precipitate the recombinant type III humanized collagen after fermentation, which is the low O-glycosylated recombinant type III humanized collagen.

[0008] By adopting the above technical scheme, during the preparation process, the metabolic rate of 6-phosphate mannose in the cells is accelerated by overexpressing phosphomannose isomerase (PMI), thereby curbing the accumulation of a large amount of 6-phosphate mannose in the cells, so as to reduce the O-glycosylation modification of type III humanized collagen. This can effectively reduce the content of recombinant type III humanized collagen modified into mannoprotein and increase the expression level of the target protein.

[0009] In certain embodiments, the amino acid sequence of the recombinant humanized type III collagen is selected from one or more of the sequences shown in SEQ ID NO. 1-4.

[0010] In certain embodiments, the nucleotide sequence of the recombinant humanized type III collagen is selected from one or more of the sequences shown in SEQ ID NOs. 7-10.

[0011] In certain embodiments, the nucleotide sequences shown in SEQ ID NOs. 7-10 are codon-optimized to obtain the corresponding nucleotide sequences. In addition, sequence elements such as restriction sites XhoI, EcoR I, and termination codons are directly added to both ends of the nucleotide sequences of SEQ ID NOs. 7-10 to obtain the final designed nucleotide sequences shown in SEQ ID NOs. 11-14, which are used for the expression of low O-glycosylated recombinant humanized type III collagen.

[0012] In certain embodiments, step 2 specifically involves transferring a recombinant plasmid overexpressing phosphomannose isomerase (PMI) into a yeast strain, and obtaining the strain through screening.

[0013] In certain embodiments, the Pichia pastoris strains include Pichia pastoris expression strains commonly used in the art, such as wild-type strain X33, histidine dehydrogenase-deficient (HIS4) strains GS115 and KM71H, and protease-deficient (pep4) strain SMD1168, and also include genetically engineered strains with Pichia pastoris expression systems obtained by those skilled in the art through experimental means. In certain embodiments, the histidine dehydrogenase-deficient (HIS4) strain GS115 is preferred.

[0014] In certain embodiments, the protein sequence of the phosphomannose isomerase (PMI) is shown as SEQ ID NO.5, and the nucleotide sequence of the phosphomannose isomerase (PMI) is shown as SEQ ID NO.6.

[0015] In certain embodiments, the preparation method further comprises step 4, purifying the low O-glycosylated recombinant humanized type III collagen obtained by fermentation: purifying with an anionic filler, collecting the eluate, and ultrafiltration, concentrating, and freeze-drying the eluate.

[0016] In a second aspect, the present application also provides a low O-glycosylated recombinant humanized type III collagen prepared by the preparation method described in the first aspect.

[0017] In a third aspect, the present application further provides a composition having cell repair or cell proliferation effects, wherein the main active ingredient of the composition comprises the low O-glycosylated recombinant humanized type III collagen described in the second aspect of the present application.

[0018] In a fourth aspect, the present application further provides the use of the low O-glycosylated recombinant humanized type III collagen described in the second aspect or the composition described in the third aspect in promoting cell repair and / or promoting cell proliferation.

[0019] In a fifth aspect, the present application also provides the use of the low O-glycosylated recombinant humanized type III collagen described in the second aspect or the composition described in the third aspect in the preparation of a product that promotes cell repair and / or promotes cell proliferation, wherein the product is a medical device, cosmetic or biopharmaceutical preparation.

[0020] Compared with the prior art, this application has the following beneficial effects: 1) The present application accelerates the rate of metabolism of 6-phosphate mannose to 6-phosphate fructose in cells by overexpressing phosphomannose isomerase (PMI) in Pichia pastoris, thereby reducing the content of 6-phosphate mannose in cells and reducing the probability of O-glycosylation modification in the Pichia pastoris expression system. The mannose-type glycopeptide in the expressed recombinant type III humanized collagen is greatly reduced. At the same time, it was also found that the low O-glycosylation modified Pichia pastoris expression system constructed by the present application increased the yield of recombinant type III humanized collagen by 2.25 times compared with the conventional Pichia pastoris expression system, and the expression amount in the fermentation broth was as high as 3.4 g / L, which is conducive to large-scale production and promotion.

[0021] 2) The low O-glycosylated recombinant type III humanized collagen sample obtained by the preparation method of the present application was subjected to circular dichroism detection, and a recombinant type III humanized collagen with a triple helix structure was successfully expressed. Ultra-high performance liquid chromatography detection also proved that the mannose glycosylation of the recombinant type III humanized collagen obtained in the present application was significantly reduced, and the mannose (Man) content was reduced by 31%-48%. After examining the migration rate and proliferation rate of 3T3 cells, it was confirmed that the low O-glycosylated recombinant type III humanized collagen prepared in the present application is safe, non-cytotoxic, and can promote cell migration and proliferation. It can be used as a high-quality raw material in medical devices, cosmetics, and biopharmaceutical preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings described herein are used to provide further understanding of the present application, constitute a part of the present application, and do not constitute an improper limitation of the present application.

[0023] Figure 1 This is the plasmid map of pPIC9k-3col001 / 3col002 / 3col003 / 3col004.

[0024] Figure 2 This is the plasmid map of pPICZαA-PMI.

[0025] Figure 3 Comparison of shake flask fermentation expression results of the strains constructed in the examples and comparative examples; wherein, lane 1: GS115 / pPICZαA-PMI / pPIC9k-3col001, lane 2: GS115 / pPIC9k-3col001, lane 3: GS115 / pPICZαA-PMI / pPIC9k-3col002, lane 4: GS115 / pPIC9k-3col002, lane 5: GS115 / pPICZαA-PM1 / pPIC9k-3col003, lane 6: GS115 / pPIC9k-3col003, lane 7: GS115 / pPICZαA-PM1 / pPIC9k-3col004, lane 8: GS115 / pPIC9k-3col004.

[0026] Figure 4 Circular dichroism spectra of target protein samples obtained in Examples and Comparative Examples.

[0027] Figure 5 This is a cell map of 3T3 cells migrating after the low O-glycosylated recombinant humanized type III collagen prepared in the example.

[0028] Figure 6 This is a bar graph showing the migration rate of 3T3 cells expressing the low O-glycosylated recombinant humanized type III collagen prepared in the example.

[0029] Figure 7 This is a data graph of the cell absorbance value of the low O-glycosylated recombinant type III humanized collagen prepared in the example. DETAILED DESCRIPTION

[0030] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed according to conventional conditions, conditions described in laboratory manuals, or conditions recommended by the manufacturer.

[0031] The present application provides a method for preparing low O-glycosylated recombinant humanized type III collagen, comprising the following steps: 1) Construction of pPIC9k-3col series recombinant plasmids In this application, the human type III collagen amino acid sequence is selected as shown in SEQ ID NO.1 / SEQ ID NO.2 / SEQ ID NO.3 / SEQ ID NO.4. These sequences have different amino acid S / T ratios, and codon optimization is performed to obtain the corresponding nucleotide sequences SEQ ID NO.7 / SEQ ID NO.8 / SEQ ID NO.9 / SEQ ID NO.10. Sequence elements such as restriction sites XhoI, EcoR I, and stop codons are directly added to both ends of these nucleotide sequences. The final designed nucleotide sequence is shown in SEQ ID NO.11 / SEQ ID NO.12 / SEQ ID NO.13 / SEQ ID NO.14. Then, SEQ ID NO.11 / SEQ ID NO.12 / SEQ ID NO.13 / SEQ ID NO.14 are ligated to the pPIC9k expression vector using restriction endonucleases XhoI, EcoR I, and T4 DNA Ligase to prepare the pPIC9k-3col series of recombinant plasmids.

[0032] 2) Construction of GS115 / pPICZαA-PMI overexpression strain The natural amino acid sequence of PMI was selected as shown in SEQ ID NO.5, and the codon-optimized nucleotide sequence was shown in SEQ ID NO.6. The PMI nucleotide sequence was ligated to the pPICZαA vector using restriction endonucleases Bsp 119I, Not I, and T4 DNA Ligase, and the α-factor signal peptide on the pPICZαA vector was removed to obtain the pPICZαA-PMI recombinant plasmid.

[0033] The plasmid was linearized using the restriction endonuclease Sac I restriction enzyme cutting site, and then the linearized plasmid was transformed into Pichia pastoris GS115 competent cells to obtain transformants. After bleomycin resistance screening, GS115 / pPICZαA-PMI positive transformants were obtained.

[0034] 3) Construction of a strain expressing low O-glycosylated recombinant humanized collagen type III The pPIC9k-3col series expression plasmids were linearized using the restriction endonuclease Sal I site, and then the linearized plasmids were transformed into the Pichia pastoris GS115 / pPICZαA-PMI strain to obtain transformants. After MD plate screening, G418 resistance screening, and yeast colony PCR identification, positive transformants of the expression strain were obtained.

[0035] 4) Shake flask expression screening of positive transformants.

[0036] The correctly identified transformants were expressed in shake flasks using BMGY and BMMY media. Fermentation was induced with methanol for 68 hours, and expression was detected by SDS-PAGE electrophoresis. Expression strains were screened and identified. Engineered strains with high expression capacity were selected for subsequent production process development.

[0037] 5) The crude protein was prepared by precipitation with ammonium sulfate solution, and then purified by anion exchange chromatography, ultrafiltration, and freeze-drying to obtain recombinant humanized type III collagen with a triple helical structure.

[0038] The following describes it in detail with reference to the embodiments.

[0039] Example 1 1. Construction of pPIC9k-3col001 recombinant plasmid 1.1 In this application, the natural human Homo sapiens collagen type III, alpha 1 chain (COL3A1, GeneBank No. NM_000090.4) sequence was selected to screen for the stable peptide 3col001, whose amino acid sequence is shown in SEQ ID NO.1, with S amino acids accounting for 8.3%. Codon optimization was performed to obtain the nucleotide sequence shown in SEQ ID NO.7. Sequence elements such as restriction sites and stop codons were then directly added to both ends of the nucleotide sequence. The final designed nucleotide sequence is shown in SEQ ID NO.11.

[0040] 6 μL of the fully synthesized T-3col001 gene and the expression vector pPIC9k were added to PCR tubes. The fragments were digested using 1 μL each of the restriction endonucleases XhoI and EcoRI, 2 μL of 10x buffer, and 10 μL of double-distilled water at 37°C in a PCR instrument for 15 minutes. The digestion reaction was performed by electrophoresis on a 1% agarose gel to obtain the target fragment and plasmid, which were then recovered using the Tiangen DNA recovery kit.

[0041] 1.2 Plasmid ligation and transformation The target fragment 3col001 obtained above was ligated with the expression vector pPIC9k at an 8:2 ratio using T4 DNA Ligase at 22°C for 2 hours. After 2 hours, 5 μL of the ligation product was mixed with 50 μL of E. coli DH5α competent cells and incubated on ice for 30 minutes. Heat transformation was then performed at 42°C for 90 seconds. The transformation solution was spread onto LB plates with the corresponding resistance (ampicillin resistance / bleomycin resistance) and incubated upside down in a 37°C incubator overnight.

[0042] 1.3 Identification of positive transformants From the above plates, positive transformants were selected and inoculated into LB liquid medium with corresponding resistance (ampicillin resistance / bleomycin resistance), cultured overnight at 37°C and 220 rpm, and plasmids were extracted using the Tiangen small extraction kit and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the designed sequence, and the correct positive transformants were saved, namely the pPIC9k-3col001 recombinant plasmid (such as Figure 1 shown).

[0043] 1.4 Linearization of recombinant plasmid Inoculate 50 mL of LB medium with the pPIC9k-3col001 recombinant plasmid and culture overnight at 37°C at 220 rpm. Extract the plasmid using the Tiangen Plasmid Miniprep Kit. Then, digest 200 μL of the plasmid with 8 μL of the restriction enzyme SalI, 60 μL of 10x buffer, and 332 μL of double-distilled water at 37°C for 20 minutes. Recover the pPIC9k-3col001 recombinant plasmid using a DNA recovery kit to complete linearization.

[0044] 2. Construction of GS115 / pPICZαA-PMI overexpression strain 2.1 Construction of PICZaA-PMI recombinant plasmid 2.1.1 Preparation of target gene The native amino acid sequence of PMI was selected as shown in SEQ ID NO. 5, and the codon-optimized nucleotide sequence was shown in SEQ ID NO. 6. 6 μL of the fragment and pPICZαA plasmid were added to PCR tubes. The fragments were digested using 1 μL each of the restriction endonucleases Bsp119I and Not I, 2 μL of 10x buffer, and 10 μL of double-distilled water at 37°C in a PCR instrument for 15 minutes. Enzymatic digestion was performed by electrophoresis on a 1% agarose gel to obtain the target fragment and plasmid, which were then recovered using the Tiangen DNA recovery kit.

[0045] 2.1.2 Plasmid ligation and transformation The target fragment (PMI) obtained above was ligated to the expression vector (pPICZαA) at an 8:2 ratio using T4 DNA Ligase at 22°C for 2 hours. After 2 hours, 5 μL of the ligation product was mixed with 50 μL of E.coLi DH5α competent cells and incubated on ice for 30 minutes. Heat transformation was then performed at 42°C for 90 seconds. The transformation solution was plated onto LB plates with the corresponding resistance (ampicillin resistance / bleomycin resistance) and incubated upside down in a 37°C incubator overnight.

[0046] 2.1.3 Identification of positive transformants From the above plates, positive transformants were selected and inoculated into LB liquid medium with corresponding resistance (ampicillin resistance / bleomycin resistance), cultured overnight at 37°C and 220 rpm, and plasmids were extracted using the Tiangen small extraction kit and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the designed sequence, and the correct positive transformants were saved, namely pPICZαA-PMI. Figure 2 shown.

[0047] 2.2 Construction of GS115 / pPICZαA-PMI strain 2.2.1 Linearization of recombinant plasmid Inoculate 50 mL of pPICZαA-PMI recombinant plasmid into LB medium and culture overnight at 37°C at 220 rpm. Extract the plasmid using the Tiangen Plasmid Miniprep Kit. Then, digest 200 μL of the plasmid with 8 μL of the restriction enzyme SacI, 60 μL of 10x buffer, and 332 μL of double-distilled water at 37°C for 20 minutes. Recover the pPICZαA-PMI using a DNA recovery kit to complete the linearization of the pPICZαA-PMI.

[0048] 2.2.2 Electroporation of recombinant plasmids and identification and screening of positive strains The linearized plasmid pPICZαA-PMI was mixed with GS115 competent cells, placed on ice for 30 min, and transformed by electroporation using an electroporator with the following parameters: voltage 2000 V, capacitance 25 μF, resistance 200 Ω. The transformation solution was spread onto cells containing bleomycin resistance and cultured at 29°C.

[0049] Transformants growing on resistant plates were identified using a yeast colony PCR kit. Colonies were picked and lysed using the lysis buffer provided in the kit. PCR was then performed using the PCR mix provided in the kit and 3'AOX / 5'AOX universal primers according to the kit's instructions. The PCR product was analyzed by electrophoresis on a 1% agarose gel. If the band size matched the theoretical size of approximately 1527 bp, the positive clone was confirmed as the GS115 / pPICZαA-PMI strain.

[0050] 3. Construction of a strain expressing low O-glycosylated recombinant humanized collagen type III The linearized recombinant plasmid pPIC9k-3col001 obtained in step 1 was transformed into the GS115 / pPICZαA-PMI strain, mixed well, and placed in an ice bath for 30 minutes. Using an electroporator, set the parameters as follows: voltage 2000V, capacitance 25μF, resistance 200Ω, and perform electroporation. The transformation solution was spread onto a plate containing G418 resistance and cultured at 29°C.

[0051] Transformants growing on resistant plates were identified using a yeast colony PCR kit. Colonies were picked and lysed using the lysis buffer provided in the kit. PCR was then performed using the PCR mix provided in the kit and 3'AOX / 5'AOX universal primers according to the kit's instructions. The PCR product was analyzed by electrophoresis on a 1% agarose gel. If the band size matched the theoretical size of 989 bp, the positive clone was confirmed as GS115 / pPICZαA-PMI / pPIC9k-3col001.

[0052] 4. Shake flask fermentation expression of low O-glycosylated recombinant humanized type III collagen in yeast transformants 4.1 Pick a single positive colony and inoculate it into a 250 mL Erlenmeyer flask containing 30 mL of BMGY medium. Incubate in a shaking incubator at 29°C and 225 rpm for 60 hours.

[0053] 4.2 Number the 50mL centrifuge tubes, pour the BMGY culture medium into the centrifuge tubes, and centrifuge at 3000 rpm for 5 minutes at room temperature to collect the shake flask bacteria.

[0054] 4.3 Add 30 mL of sterile double-distilled water to resuspend the cells. Centrifuge at 3000 rpm for 5 minutes at room temperature to collect the cells.

[0055] 4.4 Repeat step 4.3 twice.

[0056] 4.5 Collect the cells by centrifugation at 3000 rpm for 5 min at room temperature, add 30 mL of BMMY medium to resuspend the cells, pour 30 mL of the mixed BMMY culture medium into a 250 mL Erlenmeyer flask, and culture on a shaking platform at 29°C and 225 rpm.

[0057] 4.6 Add 300 μL of methanol after 24 hours of shaking culture.

[0058] 4.7 Add 300 μL of methanol after 48 hours of shaking culture.

[0059] 4.8 Add 300 μL of methanol after 72 hours of shaking culture.

[0060] 4.9 After 96 hours of shaking culture, collect the supernatant from the shake flask and store it at -20°C.

[0061] 4.10SDS-PAGE electrophoresis was used to detect the shake flask results. The band size was observed to be consistent with the theoretical size of 16.63 kDa, confirming the expression of the target protein 3col001-2.

[0062] Example 2 This example differs from Example 1 only in the peptide selected in step 1.1. In this example, the natural human Homo sapiens collagen type III, alpha 1 chain (COL3A1, GeneBank No. NM_000090.4) sequence was selected for screening for the stable peptide 3col002, whose amino acid sequence is shown in SEQ ID NO. 2, with S amino acids accounting for 8.3%. Codon optimization was performed to obtain the nucleotide sequence shown in SEQ ID NO. 8. Sequence elements such as restriction sites and stop codons were then directly added to both ends of the nucleotide sequence, resulting in the final designed nucleotide sequence shown in SEQ ID NO. 12.

[0063] The remaining steps were the same as those in Example 1, and a positive clone strain GS115 / pPICZαA-PMI / pPIC9k-3col002 was obtained. The target protein 3col002-2 (theoretical size: 16.70 kDa) was finally obtained through expression.

[0064] Example 3 This example differs from Example 1 only in the peptide selected in step 1.1. In this example, the natural human Homo sapiens collagen type III, alpha 1 chain (COL3A1, GeneBank No. NM_000090.4) sequence was selected to screen for the stabilizing peptide 3col003, whose amino acid sequence is shown in SEQ ID NO. 3 and does not contain the amino acids S / T. Codon optimization was performed to obtain the nucleotide sequence shown in SEQ ID NO. 9. Sequence elements such as restriction sites and stop codons were then added directly to both ends of the nucleotide sequence, resulting in the final designed nucleotide sequence shown in SEQ ID NO. 13.

[0065] The remaining steps were the same as those in Example 1, and a positive clone strain GS115 / pPICZαA-PMI / pPIC9k-3col003 was obtained. The target protein 3col003-2 (theoretical size: 16.63 kDa) was finally obtained through expression.

[0066] Example 4 This example differs from Example 1 only in the peptide selected in step 1.1. In this example, the natural human Homo sapiens collagen type III, alpha 1 chain (COL3A1, GeneBank No. NM_000090.4) sequence was selected for screening for the stabilizing peptide 3col004, whose amino acid sequence is shown in SEQ ID NO. 4, with T amino acids accounting for 8.3%. Codon optimization was performed to obtain the nucleotide sequence shown in SEQ ID NO. 10. Sequence elements such as restriction sites and stop codons were then directly added to both ends of the designed nucleotide sequence, resulting in the final designed nucleotide sequence shown in SEQ ID NO. 14.

[0067] The remaining steps were the same as those in Example 1, and a positive clone strain GS115 / pPICZαA-PMI / pPIC9k-3col004 was obtained. The target protein 3col004-2 (theoretical size: 17.72 kDa) was finally obtained through expression.

[0068] Example 5 The low O-glycosylated recombinant type III humanized collagen 3col001, 3col002, 3col003, and 3col004 obtained in Examples 1-4 were purified: the fermentation broth was subjected to solid-liquid separation, and the target protein was precipitated using ammonium sulfate solution (supersaturation of 16%-18%), and centrifuged. Purification was performed using a DEAE anionic filler, and the eluate was collected. The eluate was ultrafiltered using an ultrafiltration system until the retentate conductivity was less than 4mS / cm, and concentrated to a protein content greater than 1mg / mL, and the ultrafiltration was terminated. The concentrated solution concentrated by ultrafiltration was placed in a freeze dryer and freeze-dried to obtain purified protein samples 3col001-2, 3col002-2, 3col003-2, and 3col004-2.

[0069] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the pPICZαA-PMI recombinant plasmid was not constructed during the protein expression process, and the expression strain obtained was GS115 / pPIC9k-3col001. The remaining steps were the same as in Example 1, and the fermentation broth was purified by the method of Example 5 to obtain the purified protein sample 3col001-1.

[0070] Comparative Example 2 The difference between Comparative Example 2 and Example 2 is that the pPICZαA-PMI recombinant plasmid was not constructed during the protein expression process, and the expression strain obtained was GS115 / pPIC9k-3col002. The remaining steps were the same as in Example 2, and the fermentation broth was purified by the method of Example 5 to obtain the purified protein sample 3col002-1.

[0071] Comparative Example 3 The difference between Comparative Example 3 and Example 3 is that the pPICZαA-PMI recombinant plasmid was not constructed during the protein expression process, and the expression strain obtained was GS115 / pPIC9k-3col003. The remaining steps were the same as in Example 3, and the fermentation broth was purified by the method of Example 5 to obtain the purified protein sample 3col003-1.

[0072] Comparative Example 4 The difference between Comparative Example 4 and Example 4 is that the pPICZαA-PMI recombinant plasmid was not constructed during the protein expression process, and the expression strain obtained was GS115 / pPIC9k-3col004. The remaining steps were the same as in Example 4, and the fermentation broth was purified by the method of Example 5 to obtain the purified protein sample 3col004-1.

[0073] Effect Example 1 Comparison of expression levels in shake flask fermentation One strain each of GS115 / pPICZαA-PMI / pPIC9k-3col001 / 3col002 / 3col003 / 3col004 and GS115 / pPIC9k-3col001 / 3col002 / 3col003 / 3col004 was selected and expressed in shake flasks. Under the same expression conditions, the fermentation broth was subjected to electrophoresis detection. The electrophoresis patterns are shown in the figure below. Figure 3 As shown in Table 1, the protein expression levels of GS115 / pPIC9k-3col001 / 3col002 / 3col003 / 3col004 and GS115 / pPICZαA-PMI / pPIC9k-3col001 / 3col002 / 3col003 / 3col004 strains were integrated using Image J software peak area data.

[0074] Table 1 Peak area data of recombinant strains using Image J software Lane number Recombinant strain name Peak area 1 GS115 / pPICZαA-PMI / pPIC9k-3col001 52986.664 2 GS115 / pPIC9k-3col001 25364.217 3 GS115 / pPICZαA-PMI / pPIC9k-3col002 38036.551 4 GS115 / pPIC9k-3col002 22113.267 5 GS115 / pPICZαA-PMI / pPIC9k-3col003 40262.746 6 GS115 / pPIC9k-3col003 24416.924 7 GS115 / pPICZαA-PMI / pPIC9k-3col004 53594.865 8 GS115 / pPIC9k-3col004 23740.803 pass Figure 3 As shown in Table 1, the protein expression product peak area data of the GS115 / pPICZαA-PMI overexpressing strain were higher than those of the non-overexpressing strain, with GS115 / pPICZαA-PMI / pPIC9k-3col004 showing the highest expression level, significantly higher than GS115 / pPIC9k-3col004. The product peak area data also showed that the product peak area of ​​GS115 / pPICZαA-PMI / pPIC9k-3col004 was 2.25 times that of GS115 / pPIC9k-3col004. This demonstrates that intracellular overexpression of phosphomannose isomerase (PMI) can promote the expression of recombinant humanized type III collagen in GS115. The present application accelerates the metabolic rate of 6-phosphate mannose in the cell by overexpressing phosphomannose isomerase intracellularly, causing it to move towards the production of 6-phosphate fructose, thereby accelerating the sugar metabolism rate. On the one hand, it provides energy for the secretory expression of the target protein. On the other hand, 6-phosphate fructose can produce pyruvate, which is further converted and participates in the tricarboxylic acid cycle to produce intermediates such as α-ketoglutarate that can be used to synthesize proline, glycine, etc., thereby increasing the secretory expression of the target protein.

[0075] Effect Example 2: Triple Helix Structure Characterization Detection Method: The circular dichroism (CD) method commonly used in this field is used to characterize the structure of collagen. CD is a spectroscopic method used to determine the structure of compounds with chiral structures that can produce differential absorption of left and right optical rotation. It is mainly used to determine the asymmetry of molecular structures. Generally, biological macromolecules contain chiral groups and structures, so CD is often used to measure and observe changes in the structure and conformation of biological macromolecules. The CD characteristics of the triple helix structure of collagen are generally a positive absorption peak near 221nm and a negative absorption peak near 195nm. The position of the absorption peak will shift with changes in the amino acid sequence and length.

[0076] The above samples: 3col001-1, 3col002-1, 3col003-1, 3col004-1, 3col001-2, 3col002-2, 3col003-2, 3col004-2, a total of 8 samples, were prepared at room temperature and the sample concentration was 0.05 mg / mL for CD detection.

[0077] 2.3 Experimental Results The results are as follows Figure 4 As shown, 8 samples, 3col001-1, 3col002-1, 3col003-1, 3col004-1, 3col001-2, 3col002-2, 3col003-2, and 3col004-2, all have a maximum characteristic positive peak at about 221 nm, and a negative peak appears at less than 200 nm, indicating that the GS115 genetically engineered bacteria prepared by the examples of the present application successfully express recombinant type III collagen with a triple helical structure. The positive absorption peaks at approximately 221 nm for samples 3col001-2, 3col002-2, 3col003-2, and 3col004-2 were all higher than those for samples 3col001-1, 3col002-1, 3col003-1, and 3col004-1, indicating that the triple helical structure of samples obtained by intracellular overexpression of PMI was enhanced compared to that of samples obtained without intracellular overexpression of PMI. This suggests that low O-glycosylation modification is beneficial for stabilizing the triple helical structure of recombinant humanized type III collagen. The target protein obtained through this protocol exhibits fewer glycans bound to the peptide on the amino acid side chains, resulting in less steric hindrance when the recombinant protein forms a higher-order spatial structure, leading to a higher peak and a more stable triple helical structure.

[0078] Effect Example 3 Monosaccharide Composition Analysis 1. Methods: Glycosylation is a key post-translational modification of eukaryotic proteins. Glycosylation of glycoproteins is highly heterogeneous, with sugar composition and content varying across different sources. Therefore, studying the structure of oligosaccharide chains and their monosaccharide composition is crucial. Monosaccharide composition analysis of biological products can accurately determine important structural and impurity information, including the monomeric form and content of their sugar components.

[0079] 2. Experimental Instruments Ultra-high performance liquid chromatography (Waters / ACQUITY UPLC H-Class), compact thermostatic mixer (Eppendorf / ThermoMixer C), low-temperature high-speed centrifuge (Thermo / Legeno Micro 21R) 3. Data Processing Empower software (Waters) was used to integrate the chromatographic peaks in the fluorescence chromatogram to obtain the retention time and chromatographic peak area.

[0080] 4. Experimental 1) Sample pretreatment: a) Glycoprotein Monosaccharide Release: Take one PP hydrolysis tube and add 800 μL of 6 M HCl to the sample solution, shake well, and centrifuge. Simultaneously, take one monosaccharide standard, glucose (Glc) N, and add 200 μL of 6 M HCl. Place the sample and monosaccharide standard solution in a 100°C thermostat and hydrolyze for 3 hours. Methanolic Sodium Acetate / Borate Buffer: Dissolve 1.2 g of anhydrous sodium acetate and 1.0 g of boric acid in 50 mL of methanol to prepare the Derivatization Reagent Solution: Dissolve 150 mg of anthranilic acid and 99 mg of sodium cyanoborohydride in 5 L of methanolic acetic acid / boric acid buffer to prepare the Derivatization Reagent Solution. Monosaccharide Reference Standard Stock Solution: Accurately weigh 10 mg of the reference standards glucose, galactose, mannose, fucose, glucosamine, and xylose to make a monosaccharide stock solution and dilute to 10 mL. Mix equal volumes to prepare the M6 ​​standard.

[0081] b) Monosaccharide 2-AA Fluorescent Labeling: Add 50 μL of NaOAc to the dried monosaccharide standard sample, mix thoroughly by vortexing, sonicate for 15 minutes, and centrifuge for labeling. For each dried monosaccharide sample for testing and calculation, add 100 μL of derivatization reagent solution, mix thoroughly by vortexing, and briefly centrifuge. Incubate in a thermostatic heater at 80°C for 45 minutes. After labeling, remove from the sample, cool to room temperature, and store at -20°C until testing.

[0082] 5. Test results The results of the above-mentioned method are shown in Table 2.

[0083] Table 2 Analysis of monosaccharide content in samples Analysis of the data in Table 2 shows that the mannose (Man) content of the low-O-glycosylated recombinant humanized type III collagen proteins 3col001-1, 3col002-1, and 3col004-1 ranged from 43.2% to 46.6%. Despite the absence of S / T amino acids, 3col003-1 also had a mannose (Man) content of 24.6%. After expression in the GS115 / pPICZαA-PMI strain, the mannose (Man) content of 3col001-2, 3col002-2, 3col003-2, and 3col004-2 was significantly reduced, by 31% to 48%. This reduction was also evident in 3col003-2, which lacks S / T amino acids. Furthermore, the galactose (Gal), glucose (Glc), and fucose (Fucose) contents were significantly reduced, reducing the immunogenicity of the proteins. Since O-glycosylation plays a major role in the expression of collagen, and mannose-type glycoproteins can cause some immune responses, reducing O-glycosylation modification in the yeast expression system can effectively reduce the immunogenic response.

[0084] Effect Example 4: Cell scratch test with low O-glycosylated recombinant humanized type III collagen 1. Methods: Laying plate: 2×10 6 The number of cells was inoculated into a 6-well plate, and after culturing for 24 hours (adjustable), the cells were streaked with a pipette tip and washed three times with PBS.

[0085] Then, 0.05 mg / mL of 3col001-2, 3col002-2, 3col003-2, and 3col004-2 protein samples were added to serum-free medium for incubation, and a blank control containing only serum-free medium was set up. Pictures were taken under a microscope at 0, 24, 48, and 72 hours of incubation. Figure 5 shown.

[0086] The photos were analyzed using Image J and Prism 8, and cell migration rate and time histograms were drawn, as shown in the following figure. Figure 6 (P≤0.01) (“*” P<0.05, significant difference; “**” P<0.01, extremely significant difference).

[0087] pass Figure 5 and Figure 6 It can be seen that the low O-glycosylated recombinant humanized type III collagen obtained in the present application can significantly promote cell migration at a concentration of 0.05 mg / ml and has a strong tissue repair ability.

[0088] Effect Example 5: Absorbance Test Experiment on the Promotion of Cell Proliferation by Recombinant Humanized Type III Collagen with Low O-Glycosylation 1. Method: Planking: Place 10 3 Cells were plated into 96-well plates (7 plates total) at a concentration of 100 cells / well. After culturing for 24 hours (adjustable), 3col001-2, 3col002-2, 3col003-2, and 3col004-2 were added at a concentration of 0.05 mg / mL in 5% serum medium. A blank control group was incubated with only 5% serum medium. Subsequently, the cells were incubated in an incubator, and one plate was sampled every 24 hours for MTT assay.

[0089] The cell status was recorded using an inverted imaging microscope, and then the culture medium was removed and 50 μL of MTT solution was added to each well and incubated at 37°C in 5% CO2 for 2 h.

[0090] The MTT solution was then removed, and 100 μL of isopropanol was added to each well and shaken at room temperature for 30 min. The absorbance of each well was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA).

[0091] GraphPad Prism 8 was used to analyze the absorbance data of protein samples, and a line graph of the absorbance (OD) values ​​and time of samples 3col001-2, 3col002-2, 3col003-2, and 3col004-2 was drawn, as shown in Figure 1. Figure 7 shown.

[0092] pass Figure 7 It can be seen that 3col001-2, 3col002-2, 3col003-2, and 3col004-2 at a concentration of 0.05 mg / mL can all significantly promote cell proliferation, among which 3col004-2 has the best effect in promoting cell proliferation at a concentration of 0.05 mg / ml.

[0093] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for preparing low O-glycosylated recombinant humanized type III collagen, characterized in that: The preparation method The following steps are included: Step 1, obtaining a recombinant plasmid expressing recombinant humanized type III collagen; Step 2, obtaining a strain overexpressing phosphomannose isomerase (PMI); Step 3: linearize the recombinant plasmid described in step 1 and transfer it into the strain described in step 2, ferment and culture it, and precipitate the recombinant type III humanized collagen after fermentation, which is the low O-glycosylated recombinant type III humanized collagen.

2. The preparation method according to claim 1, characterized in that The amino acid sequence of the recombinant humanized type III collagen is selected from one or more sequences shown in SEQ ID NO. 1-4.

3. The preparation method according to claim 1, characterized in that The nucleotide sequence of the recombinant humanized type III collagen is selected from one or more sequences shown in SEQ ID NOs. 7-10.

4. The preparation method according to claim 1, characterized in that Specifically, step 2 involves transferring a recombinant plasmid that overexpresses phosphomannose isomerase (PMI) into Pichia pastoris, and obtaining the strain through screening.

5. The preparation method according to claim 1, characterized in that The protein sequence of the phosphomannose isomerase (PMI) is shown in SEQ ID NO.5, and the nucleotide sequence of the phosphomannose isomerase (PMI) is shown in SEQ ID NO.

6.

6. The preparation method according to claim 1, characterized in that The preparation method further comprises step 4, purifying the low O-glycosylated recombinant humanized type III collagen obtained by fermentation: purifying with an anionic filler, collecting the eluate, and ultrafiltration concentrating and freeze-drying the eluate.

7. Low O-glycosylated recombinant humanized type III collagen prepared by the preparation method according to any one of claims 1 to 6.

8. A composition having cell repair or cell proliferation properties, characterized in that: The main active ingredient of the composition comprises the low O-glycosylated recombinant type III humanized collagen according to claim 7.

9. Use of the low O-glycosylated recombinant humanized type III collagen according to claim 7 or the composition according to claim 8 in promoting cell repair and / or cell proliferation.

10. Use of the low O-glycosylated recombinant humanized type III collagen according to claim 7 or the composition according to claim 8 in the preparation of a product for promoting cell repair and / or cell proliferation, characterized in that: The product is a medical device, cosmetic or biopharmaceutical preparation.

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