A recombinant keratin K35, a pichia pastoris expression vector and construction method thereof, and a eukaryotic expression method thereof

Abstract

The present application relates to the technical field of recombinant protein, and particularly relates to screening of a recombinant keratin K35 mutant, construction of a Pichia pastoris expression vector and protein expression. The recombinant keratin K35M has an amino acid sequence as shown in SEQ ID NO. 2. The wild-type keratin K35 is subjected to sequence optimization through site-directed mutation, and the mutation site is determined as M237L / S243N, and part of the amino acids at the N-terminal and C-terminal are removed, and the central alpha helix rod-shaped region is reserved. The binding capacity of the protein to calcium ions is improved through the above modification, and the stability, functional activity and expression level of the protein itself are improved, which lays a molecular foundation for the application of the protein in tissue repair, oral cavity repair and the like. The technical scheme can solve the problems of the existing recombinant keratin K35, such as insufficient calcium chelation capacity, low recombinant expression level and disadvantages of a prokaryotic expression system, and significantly improves the application value and industrialization potential of the recombinant keratin.

Description

Technical Field

[0001] This invention relates to the field of recombinant protein technology, specifically to a recombinant keratin K35 and its Pichia pastoris expression vector and construction method, as well as its eukaryotic expression method. Background Technology

[0002] Keratin is a class of structural biomolecules widely distributed in nature, mainly found in animal fur, feathers, horns, hooves, and human hair and nails, where it is a major structural component. As a natural biomolecule, keratin possesses excellent bioactivity, good biocompatibility, and biodegradability, while also exhibiting outstanding material mechanical properties and abundant natural availability. Its application in the biomedical field has a long history, and it has gradually become a hot material for interdisciplinary research, showing broad application prospects in tissue engineering, hemostasis, cell culture, anti-inflammation, and many other areas, demonstrating significant biological and biomedical application potential.

[0003] Currently, keratin is mainly obtained through two methods: traditional extraction and recombinant genetic engineering. Traditional extraction uses animal-derived materials (such as wool, feathers, and human hair) as raw materials, obtaining keratin through physicochemical degradation and purification processes. However, this method suffers from drawbacks such as low extraction efficiency, uneven product purity, and easy destruction of the protein's natural structure. Furthermore, the source of raw materials is limited by natural resources, making it difficult to meet the needs of large-scale, standardized applications. It also carries potential biosafety risks and ethical controversies, limiting its application in high-end biomedical fields. To overcome the shortcomings of traditional extraction methods, recombinant keratin technology has emerged. Recombinant keratin is produced through genetic engineering techniques (such as microbial fermentation). Its amino acid sequence and spatial structure can be precisely designed and controlled, allowing for targeted optimization of its biological functions. The product has high purity and stable quality, enabling large-scale production and effectively compensating for the shortcomings of traditional keratin extraction. Currently, the main expression host for recombinant keratin is Escherichia coli. However, when recombinant keratin is expressed in prokaryotic cells, it is prone to forming inclusion bodies, which leads to reduced protein activity. Furthermore, the process requires bacterial disruption, which not only increases the difficulty and cost of subsequent protein purification but may also damage the protein structure, further affecting its application.

[0004] Pichia pastoris, a recognized safe microorganism that does not produce endotoxins, has successfully expressed recombinant proteins that have been used in the production of human vaccines, therapeutic proteins, and diagnostic reagents, making it an ideal eukaryotic expression host. This expression system is technologically mature, facilitates high-density fermentation, and can achieve highly efficient secretory expression and maximized yield of specific keratins through precise promoter engineering, signal peptide optimization, and multi-copy site-directed integration strategies. This lays a solid foundation for the industrial application of recombinant keratins and represents a preferred solution to overcome the shortcomings of prokaryotic expression systems.

[0005] Recombinant keratin K35, as an important member of the keratin family, has attracted widespread attention for its potential applications in biomedicine and materials science. However, existing recombinant keratin K35 technologies still suffer from significant functional and expression defects, severely limiting its industrial application and promotion. On the one hand, at the functional level, existing recombinant keratin K35 exhibits insufficient calcium chelation capacity. Calcium chelation is a key function for keratin in tissue repair and biomaterial preparation; insufficient calcium chelation makes it difficult to form stable complexes and exert corresponding effects, greatly limiting its application in dental restoration, bone tissue engineering, and related fields. On the other hand, at the engineering expression level, the expression levels of existing recombinant keratin K35 are generally unsatisfactory, especially when secreted in Pichia pastoris. Since the secretory expression process in Pichia pastoris involves multiple complex steps such as protein folding, modification, and transport, existing recombinant keratin K35 suffers from low secretory expression efficiency, resulting in product yields that cannot meet the needs of large-scale production. This also increases the cost and difficulty of the production process, further restricting its practical application.

[0006] Therefore, given the technical shortcomings of existing recombinant keratin K35, such as insufficient calcium chelating capacity, low engineered expression level, and especially low secretory expression efficiency in Pichia pastoris, developing a recombinant keratin K35 that can achieve high-level secretory expression and optimized calcium chelating function is of great practical significance and industrial value, and is also a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a recombinant keratin K35 to solve the technical problems of insufficient calcium chelating ability and low engineered expression level of existing recombinant keratin K35.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A recombinant keratin K35M, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0009] This technical solution also provides an expression vector for recombinant keratin K35M, which is formed by integrating the nucleotide fragment corresponding to recombinant keratin K35M into the multiple cloning site of an empty vector.

[0010] Furthermore, the nucleotide fragment corresponding to recombinant keratin K35M is a nucleotide fragment optimized according to the codon preference of the protein expression host.

[0011] Furthermore, the protein expression host is Escherichia coli or yeast.

[0012] Furthermore, the empty carrier is pET-28a(+) or pPICZαA.

[0013] This technical solution also provides a Pichia pastoris expression system for recombinant keratin K35M, which includes Pichia pastoris transformed with an expression vector for recombinant keratin K35M.

[0014] Furthermore, the Pichia pastoris expression system for recombinant keratin K35M is prepared by the following method: integrating a nucleotide fragment with a sequence such as SEQ ID NO. 3 into the multiple cloning site of pPICZαA to obtain an expression vector; after linearization, the expression vector is electroporated into competent Pichia pastoris cells, and then subjected to resistance screening and PCR verification to obtain the Pichia pastoris expression system.

[0015] Furthermore, the Pichia pastoris expression system was cultured in shake flasks until OD was reached after inoculation. 600 The value was 6-8; then methanol was used to induce the expression of recombinant keratin K35M; after the expression was induced, the liquid part was separated by solid-liquid separation, and the liquid part was enriched with recombinant keratin K35M.

[0016] This technical solution also provides the application of recombinant keratin K35M in the preparation of calcium ion chelating agents or oral repair preparations.

[0017] The technical principle behind this solution is: This invention focuses on the molecular modification of recombinant keratin K35M, the construction of Pichia pastoris expression vectors, and eukaryotic secretory expression. Addressing the shortcomings of existing recombinant keratin K35 technologies, such as insufficient calcium chelation function and low prokaryotic expression levels, this invention achieves a dual enhancement of recombinant protein function and expression level through molecular design and expression system optimization, as detailed below: The amino acid sequence of wild-type keratin K35 was optimized using site-directed mutagenesis, and the optimal mutation site was determined to be M237L / S243N. In addition, some amino acids in the N-terminal and C-terminal non-helical regions of wild-type keratin K35 were removed, retaining the central α-helical rod region. Through these modifications, recombinant keratin K35M (amino acid sequence shown in SEQ ID NO. 2) was obtained. This mutation and truncation design alters the amino acid sequence of wild-type K35, exposing more active sites involved in calcium chelation, enhancing its binding capacity to calcium ions, while further reducing the protein molecular weight to form a stable monomeric form. This improves the protein's stability, functional activity, and expression level, laying the molecular foundation for its application in tissue repair, oral restoration, and other scenarios.

[0018] Based on the codon usage preferences of Pichia pastoris, the nucleotide sequence corresponding to recombinant keratin K35M was optimized. This optimized nucleotide fragment was integrated into the multiple cloning site of the empty Pichia pastoris vector pPICZαA to construct the recombinant expression vector K35M-pPICZαA. A bleomycin resistance gene was introduced into the vector as a selection marker to facilitate rapid screening and identification of positive transformants. Simultaneously, the pPICZαA vector's own signal peptide guides the extracellular secretion of the recombinant protein, avoiding the drawbacks of lysis treatment in prokaryotic expression.

[0019] After linearization, the constructed recombinant expression vector was introduced into Pichia pastoris competent cells via electroporation. Screening combined with PCR verification yielded a stable Pichia pastoris expression system integrating the K35M gene. This system utilizes Pichia pastoris as the chassis cell, driving efficient expression of the recombinant K35M gene through methanol induction. Simultaneously, leveraging the advantages of the Pichia pastoris expression system, the recombinant protein achieves correct folding and modification, and is secreted extracellularly via signal peptide mediation, realizing highly efficient secretory expression.

[0020] After mutation optimization, the amino acid sequence of recombinant keratin K35M can effectively chelate calcium ions to form a stable complex. This recombinant keratin can guide the directional arrangement of calcium ions and phosphate groups to achieve biomimetic mineralization of dentin, thereby realizing functions such as oral repair. This principle also provides support for its application in calcium ion chelating agents and oral repair preparations.

[0021] Compared with existing technologies, this invention has significant advantages in recombinant protein function, expression efficiency, production process, and application scope. Specific beneficial effects are as follows: (1) Significantly enhances the calcium chelation function of recombinant keratin This invention, through site-directed mutagenesis and truncation, yields recombinant keratin K35M, which exhibits significantly enhanced calcium chelating capacity compared to wild-type K35. It can stably bind calcium ions and form complexes, effectively addressing the technical shortcomings of existing recombinant K35, such as insufficient calcium chelating capacity and difficulty in meeting the needs of tissue repair and other applications. Combined with its binding properties with hydroxyapatite, it can be widely used in calcium ion chelating agents and oral repair formulations, particularly in dental restoration and bone tissue engineering, filling a gap in the application of recombinant keratin in these high-end fields.

[0022] (2) Optimize the secretory expression efficiency of Pichia pastoris and achieve large-scale production. This invention optimizes the K35M nucleotide sequence using Pichia pastoris codon preference and leverages the signal peptide guidance of the pPICZαA vector to enable efficient extracellular secretion of recombinant K35M with high expression levels and strong protein stability. This effectively overcomes the technical barrier of low expression levels of recombinant K35 in existing E. coli expression systems. Furthermore, the Pichia pastoris expression system allows for high-density fermentation, enriching the target protein from the culture supernatant without lysis, significantly simplifying subsequent purification processes, reducing production difficulty and costs, and avoiding protein structure damage during lysis, thus ensuring the activity of the recombinant protein and meeting the needs of large-scale, standardized production. This lays a solid foundation for the industrial application of recombinant keratin K35M.

[0023] (3) Obtain new recombinant keratin varieties with superior performance Recombinant keratin K35M is an M237L / S243N double mutant with a smaller molecular weight than wild-type K35. It exists in a stable monomeric form. Compared with existing recombinant K35, its protein stability and expression uniformity are significantly improved, and it is less likely to form aggregates or denature, further ensuring its functional activity and application effect. It is a new and superior recombinant keratin biomaterial.

[0024] (4) Improve product safety and applicability This invention uses Pichia pastoris as the expression host. Pichia pastoris is a recognized safe microorganism that does not produce endotoxins. The recombinant protein expressed by Pichia pastoris is free from endotoxin contamination, resulting in higher safety. This makes it suitable for fields such as biomedicine and dental restoration, where high purity and safety are required. It effectively avoids the drawbacks of E. coli prokaryotic expression systems, such as low protein activity, the need for bacterial disruption, and the risk of endotoxin contamination. Furthermore, the Pichia pastoris expression system combines the ease of cultivation of prokaryotes with the post-translational modification capabilities of eukaryotes, making the spatial conformation of recombinant K35M closer to that of natural keratin, further enhancing its biocompatibility and bioactivity.

[0025] (5) The construction method is simple and efficient. The methods for constructing Pichia pastoris expression vectors, preparing Pichia pastoris expression systems, and eukaryotic expression methods provided by this invention are clear in steps and simple in operation. They do not require complex experimental equipment and techniques. Positive transformants can be obtained quickly through resistance screening and PCR verification. The culture conditions are easy to control, and the recombinant protein can be induced and expressed rapidly. This makes it easy for those skilled in the art to master and promote its application, and reduces the difficulty of technology implementation.

[0026] (6) To make up for the shortcomings of traditional extraction methods and existing recombination technologies Compared to traditional keratin extraction methods, this invention uses genetic engineering technology to prepare recombinant K35M, resulting in products with high purity and stable quality. It is not limited by natural resources and has no potential biosafety risks or ethical controversies. Compared to existing recombinant K35 technologies, this invention solves the defects in function and expression efficiency, significantly enhances the application value and industrialization potential of recombinant keratin, and promotes the expansion of recombinant keratin applications in biomedicine, materials science, and other fields, thus possessing significant industrial value and practical significance. Attached Figure Description

[0027] Figure 1 The following are the SDS-PAGE detection results of prokaryotic expression of different proteins in Example 3 of this invention: The leftmost image shows the marker; 1-3 represent the SDS-PAGE results of engineered bacteria transfected with the pET-28a(+)-K35M (M237L / S243N) expression vector without induction, after induction, and after induction (supernatant); 4-6 represent the SDS-PAGE results of engineered bacteria transfected with the pET-28a(+)-K35C expression vector without induction, after induction, and after induction (supernatant).

[0028] Figure 2 This is the standard curve for Embodiment 4 of the present invention.

[0029] Figure 3 The results of SDS-PAGE analysis of the Pichia pastoris protein expression induced in Example 5 of the present invention are shown (lane 1 represents the blank control, and lanes 2-3 represent the integrated Pichia pastoris strains expressing the target protein obtained in Example 5). Detailed Implementation

[0030] The present invention is further illustrated below with reference to embodiments. It should be understood that the following embodiments are for explanation and illustration only and do not limit the scope of the present invention in any way. Experimental reagents not specifically described in this invention are all conventional reagents in the art, which can be prepared according to conventional methods in the art or purchased from relevant reagent suppliers; experimental methods not specifically described are all conventional methods in the art, and relevant experimental manuals, such as the Molecular Cloning Experimental Manual or the instructions of relevant reagent manufacturers, can be consulted.

[0031] Example 1: K35M sequence information The amino acid sequence of wild-type keratin K35 is shown in SEQ ID NO. 1: MASKCLKAGFSSGSLKSPGGASGGSTRVSAMYSSSSCKLPSLSPVARSFSACSVGLGRSSYRATSCLP ALCLPAGGFATSYSGGGGWFGEGILTGN EKETMQSLNDRLAGYLEKVRQLEQENASLESRIREWCEQQVPYMCPDYQSYFRTIEELQKKTLCSKAENARLVVEIDNAKLAADDFRTKYETEVSLRQLVESDINGLRRILDDLTLCKSDLEAQVESLKEELLCLKKNHEEEVNSLRCQLGDRLN VEVDAAPPVDLNRVLEEMRCQYETLVENNRRDAEDWLDTQSEELNQQVVSSSEQLQSCQAEIIELRRTVNALEIELQAQHSMRDALESTLAETEARYSSQLAQMQCMITNVEAQLAEIRADLERQNQEYQVLLDVRARLECEINTYRGLLESEDSK LPCNPCAPDYSPSKSCLPCLPAASCGPSAARTNCSPRPICV PCPGGRF .

[0032] This technical solution modifies the sequence of wild-type keratin K35, naming it K35M (M237L / S243N). The modified amino acid sequence is shown in SEQ ID NO. 2. EKETMQSLNDRLAGYLEKVRQLEQENASLESRIREWCEQQVPYMCPDYQSYFRTIEELQKKTLCSKAENARLVVEIDNAKLAADDFRTKYETEVSLRQLVESDINGLRRILDDLTLCKSDLEAQVESLKEELLCLKKNHEEEVNSLRCQLGDRLNVEVDAAPPVDLNRVLEEMRCQY ETLVENNRRDAEDWLDTQSEELNQQVVSSSEQLQSCQAEIIELRRTVNALEIELQAQHSL (M237L) RDALEN (S243N) TLAETEARYSSQLAQMQCMITNVEAQLAEIRADLERQNQEYQVLLDVRARLECEINTYRGLLESEDSKDEEENDQVKDEEENDQVKGGGGSHHHHHH.

[0033] The difference between K35M (M237L / S243N) and K35 is: (a) K35M (M237L / S243N) is based on K35, with some amino acids removed from the non-helical regions at the N and C ends, while retaining the central α-helical rod region, as shown in the underlined sequence in SEQ ID NO. 1.

[0034] (b) After truncating the C-terminus of K35, the sequence “DEEENDQVKDEEENDQVK” (a short peptide with calcium chelation effect) was added to the C-terminus of the protein, and the histidine tag “HHHHHH” was linked to the C-terminus through the linker “GGGGS”.

[0035] (c) The 237th amino acid of K35M (M237L / S243N) is mutated from M to L (M237L) of wild-type K35; the 243rd amino acid of K35M is mutated from S to N (S243N) of wild-type K35, see the bold text in SEQ ID NO. 2 for details.

[0036] Example 2: Construction of expression vector This protocol utilizes K35M (M237L / S243N) for both prokaryotic and eukaryotic expression, with *Escherichia coli* and *Pichia pastoris* as host cells, respectively. The expression vectors used are pET-28a(+) (for prokaryotic expression) and pPICZαA (for eukaryotic secretory expression), respectively. Both empty vectors are commercially available, for example, from Invitrogen. The expression vectors were constructed by Nanjing Genscript Biotech Co., Ltd. using conventional methods. The codon-optimized nucleotide fragment corresponding to K35M (M237L / S243N) was inserted into the multiple cloning site of the empty vector pPICZαA via EcoRI and SalI restriction enzyme sites, resulting in the pPICZαA-K35M (M237L / S243N) expression vector. Similarly, the codon-optimized nucleotide fragment corresponding to K35M (M237L / S243N) was inserted into the multiple cloning site of the empty vector pET-28a(+) through two restriction enzyme sites, Ncol1 and Xhol1, and protective bases were added to prevent frameshift mutations, resulting in the pET-28a(+)-K35M(M237L / S243N) expression vector. Both the pPICZαA-K35M(M237L / S243N) and pET-28a(+)-K35M(M237L / S243N) expression vectors can be transformed into E. coli TOP10 for preservation.

[0037] The K35M (M237L / S243N) protein identified in this technical solution is obtained by modifying wild-type K35. Before attempting double-point mutations of K35M (M237L / S243N), the inventors conducted other experiments. For example, they also attempted to truncate wild-type K35 both forwards and backwards, and to add an additional peptide to the C-terminus, aiming to increase the protein's expression level in engineered bacteria and enhance its calcium-chelating ability. The protein used in these experiments is abbreviated as K35C, and its specific sequence is shown in SEQ ID NO. 3: EKETMQSLNDRLAGYLEKVRQLEQENASLESRIREWCEQQVPYMCPDYQSYFRTIEELQKKTLCSKAENARLVVEIDNAKLAADDFRTKYETEVSLRQLVESDINGLRRILDDLTLCKSDLEAQVESLKEELLCLKKNHEEEVNSLRCQLGDRLNVEVDAAPPVDLNRVL EEMRCQYETLVENNRRDAEDWLDTQSEELNQQVVSSSEQLQSCQAEIIELRRTVNALEIELQAQHSMRDALESTLAETEARYSSQLAQMQCMITNVEAQLAEIRADLERQNQEYQVLLDVRARLECEINTYRGLLESEDSKDEEENDQVKDEEENDQVKGGGGSHHHHHH.

[0038] The difference between K35C and wild-type K35 is that some amino acids in the non-helical regions of the N-terminus and C-terminus are removed; the sequence "DEEENDQVKDEEENDQVKGGGGSHHHHHH" is added to the C-terminus. The difference between K35C and K35M (M237L / S243N) in this scheme is that point mutations of M237L / S243N were not performed.

[0039] The eukaryotic expression vector for the K35C protein is the pPICZαA-K35C expression vector. It is obtained by optimizing the K35C protein sequence (SEQ ID NO. 3) using standard codons, and then inserting its nucleotide fragment into the multiple cloning site of the empty vector pPICZαA via EcoRI and SalI restriction enzyme sites. Similarly, the prokaryotic expression vector for K35C, pET-28a(+)-K35C, is obtained.

[0040] Example 3: Prokaryotic expression of protein Expression vectors (pET-28a(+)-K35C expression vector and pET-28a(+)-K35M (M237L / S243N) expression vector) were transformed into *E. coli* BL21(DE3) / Rosetta(DE3) using conventional methods. The water bath temperature was set to 42°C and preheated to the preset temperature. *E. coli* competent cells were thawed on ice, and 10 μl of the expression vector was added to 100 μl of competent cells and mixed well. The mixture was then placed on ice for 30 min. The cells were then heat-shocked in a 42°C water bath for 90 s and quickly transferred to ice for 5 min. 500 μl of LB medium (without antibiotics) was added to the competent cells containing the transformed fragment in a sterile workbench, and the mixture was incubated at 37°C for 1 h at low speed. The mixture was centrifuged at low speed, and some of the supernatant was discarded. The suspension was evenly spread on LB-K solid medium and incubated upside down in a 37°C incubator for 12–16 h. Single colonies were selected for verification. For each transformant, 3-5 single colonies were selected for colony PCR verification and sequencing. Those that were correctly verified could be preserved as expression strains. All three expression vectors were transformed into competent cells using the same method.

[0041] After screening and plate culture, qualified bacterial strains were selected, and single colonies of uniform size were inoculated into 10 mL of LB liquid medium (50 mL Erlenmeyer flask) containing a final concentration of 100 μg / mL Amp. The culture was then incubated overnight at 37°C and 200 rpm (for seed preservation). Finally, a 1% inoculum was transferred to 10 mL of LB liquid medium containing a final concentration of 50 μg / mL kana. When the bacterial concentration OD... 600 When the concentration reached 0.7, IPTG solution was added to a final concentration of 0.5 mmol / L, and the cells were cultured at 25°C and 200 rpm for 24 h. After centrifugation at 8000 rpm for 5 min at 4°C, the cells were resuspended in 10×PBS buffer to obtain a bacterial suspension. The suspension was then sonicated on ice at 75% power (total power 500 W) for 3 seconds followed by a 6-second interval, for a total duration of 10 min, to obtain bacterial lysate. The suspension was then centrifuged at 8000 rpm for 15 min at 4°C, and the supernatant and precipitate were collected for protein expression detection. Engineered bacteria transfected with the pET-28a(+)-K35C expression vector and engineered bacteria transfected with the pET-28a(+)-K35M (M237L / S243N) expression vector were used, and induced expression and uninduced expression groups were set up respectively. The induced expression group was cultured according to the above method, and the uninduced group was cultured according to the above method but without the addition of IPTG during the culture process.

[0042] The specific implementation method is as follows: After induction of expression, after centrifugation, the same weight of wet bacterial cells from each group were added to the same volume of conventional buffer to suspend the cells. The bacterial suspension was then subjected to the aforementioned sonication to obtain bacterial lysate, which was then centrifuged again to obtain supernatant and precipitate. The precipitate was resuspended in buffer of the same volume as the supernatant. Equal volumes of supernatant and precipitate resuspended were taken, and an equal volume of SDS loading buffer was added according to existing conventional methods. After mixing, the mixture was incubated at 95°C for 10 minutes. Because the sampling method described above allows the band intensity in the Coomassie Brilliant Blue results of the protein gel to directly reflect the protein expression level, it can be used to compare the expression level and solubility differences of the target protein obtained under different conditions.

[0043] In SDS-PAGE analysis, remove the comb from the gel, and using a 20µL pipette tip, pipette 10µL of the boiled sample and spot it onto the wells. Connect the positive and negative electrodes to the electrophoresis apparatus and turn on the power. The stacking gel is set to 80V for 20 minutes, and the separating gel to 160V for 40 minutes. Stop electrophoresis once all the blue color has evaporated; this typically takes 1-2 hours. Remove the gel from the electrophoresis apparatus, pry it open with a blade, and cut off the stacking gel portion. Place the gel in Coomassie Brilliant Blue R-250 staining solution at 80℃, 80 rpm for 20 minutes. Then, destain the stained protein gel in destaining solution at 80℃, 70 rpm for 60 minutes, until the background turns white. Transfer the protein gel to a gel imaging system for photographic analysis. See [link to experimental results]. Figure 1 .

[0044] Figure 1 In the image, due to the presence of irrelevant lanes between lanes 1-3 and lanes 4-6, the images of these irrelevant lanes have been hidden to facilitate a direct comparison of the expression levels of the two protein groups. Lanes 4-6 have also been shifted to the left and spliced ​​together. SDS-PAGE analysis showed that, compared to the engineered bacteria transfected with the K35C expression vector, the engineered bacteria transfected with the M237L / S243N double mutant K35M expression vector showed high expression of the target protein, producing a clear protein band around 38 kDa. The engineered bacteria transfected with the K35C expression vector did not show a clear protein band around 38 kDa, indicating that the target protein could not be expressed in large quantities. These experimental data demonstrate that the M237L / S243N mutation can effectively increase the prokaryotic expression level of K35 protein, providing an optimized solution for the large-scale prokaryotic preparation of K35 protein.

[0045] Example 4: Calcium chelation capacity test The pET-28a(+)-K35C and pET-28a(+)-K35M (M237L / S243N) engineered bacteria constructed in Example 3 were used for induced expression and cell lysis as shown in Example 3. Since both target proteins are histidine-tagged, the His-tagged fusion proteins were purified using conventional Ni-NTA affinity chromatography on the two bacterial cell lysates. After obtaining the purified K35C and K35M (M237L / S243N) proteins, the concentrations of the two purified proteins were accurately determined using the BCA method. Using the lower concentration group as a baseline, the high concentration group was diluted with buffer (20mM Tris-HCl, 150mM NaCl) to a level essentially identical to the low concentration group, and the concentration deviation between the two groups was confirmed to be less than 5% using the BCA method. Through the above operations, the concentrations of the two protein solutions were adjusted to be essentially identical to ensure that the working concentrations of the functional components (target proteins) in the two calcium chelation capacity test experiments were essentially consistent. The above adjustment steps are necessary for controlling variables and ensure that the two sets of experiments are as close as possible and comparable. According to the volume ratio of purified protein solution to calcium working solution = 1:2, CaCl2 working solution (5 mM / L) was injected into the reaction system. The system was placed in a 37℃ constant temperature shaking incubator and shaken at 150 rpm for 120 min. Then, anhydrous ethanol (9 times the volume) was added to the reaction solution, mixed well, and allowed to stand for 2 h to promote the precipitation of the protein-calcium complex. The reaction solution was centrifuged at 10000 rpm for 15 min, and the supernatant was retained to determine its calcium ion concentration.

[0046] Dilute the CaCl2 working solution (5 mM / L) according to Table 1 below and measure its OD value at 575 nm. Prepare a standard curve for calcium ion concentration. Add 2 mL of colorimetric reagent sequentially to the above standard solution and the sample to be tested (lysis buffer), mix well, let stand at room temperature for 10 min, zero the spectrophotometer with a blank tube, and measure the OD value at 575 nm. The calcium ion mass concentration is verified according to the Solarbio Calcium Assay Kit instructions (o-cresolphthalein complex ketone colorimetric method) BC8333. OD values ​​are used as the OD value. 575 The calcium content in the supernatant is calculated using the standard curve, and then the calcium ion chelation rate is calculated. The formula is: Calcium ion chelation rate = (1 - Calcium content in supernatant / Total calcium content in reaction solution) × 100%. See the standard curve for details. Figure 2 The absorbance (OD) of the two experimental groups at 575 nm was measured. 575The calcium ion content in the supernatant was calculated based on the standard curve, and then substituted into the aforementioned calcium ion chelation rate formula to calculate the calcium ion chelation rate of the two samples. The two samples were K35M (M237L / S243N) mutant protein and K35C protein, respectively. The concentrations of the purified protein solutions in both groups were adjusted to be consistent before testing to ensure good comparability of the experimental results. Therefore, the calcium ion chelation rate test results of the two protein solutions can directly reflect the difference in calcium chelation activity between K35M (M237L / S243N) and K35C proteins. Using the actual calcium ion chelation rate of the K35C protein expressed by pET-28a(+)-K35C as a benchmark and set at 100% (relative calcium ion chelation rate), the relative calcium ion chelation rates of other experimental groups were calculated. Based on the measured values ​​of the pET-28a(+)-K35M (M237L / S243N) protein solution, its relative calcium ion chelation rate was calculated to be 119.872814%. The test results are shown in Table 2, indicating that K35M improved the chelation rate by 20% compared to K35C. The double-point mutation not only increased protein expression levels but also improved the calcium ion chelation rate, effectively enhancing protein performance. Next, this experiment optimized the codons of K35M according to the codon preferences of yeast to construct a Pichia pastoris expression system.

[0047] Table 1: Dilution gradient settings

[0048] Table 2: Chelation Rate Test Results

[0049] Example 5: Construction and Induction of Pichia pastoris Expression System Different bacterial strains containing the target gene were streaked onto LB+Z resistance (Zeocin) plates corresponding to their respective resistance levels and incubated overnight at 37°C. Single clones from the overnight culture were picked and inoculated into 10 mL of LB+Z resistance liquid medium and incubated overnight at 37°C. The overnight culture was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the bacterial cells were collected for plasmid extraction. The extracted plasmid (expression vector) was measured for concentration using NanoDrop and then frozen at -20°C. Specifically, the expression vector was the pPICZαA-K35M expression vector from Example 2.

[0050] Take 20 μg of the extracted plasmid and digest it with PmeI at 37℃ for 3-6 h. Take 5 μL of the linearized gene fragment and confirm complete linearization by 1% agarose gel electrophoresis. Use the Kangwei Universal DNA Purification and Recovery Kit for liquid recovery. The specific steps are as follows: Take the completely linearized mixture and add an equal volume of PC solution from the kit. Mix well and transfer to a BL-equilibrated collection column from the kit. Centrifuge at 12000 rpm for 1 min. Wash twice with PW solution from the kit and centrifuge at 12000 rpm for 2 min. Transfer the collection column to a clean 1.5 mL centrifuge tube, air dry at room temperature for 5 min, add preheated double-distilled water at 55℃, let stand at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min to obtain the linearized gene fragment.

[0051] Add 10 μL of the linearized fragment to 100 μL of Pichia pastoris competent cells (Pichia pastoris X33), mix well on ice, and transfer to an electrode cup. Electrolyze at 1500 V and 200 mA. Immediately after electrolysis, add 1000 μL of recovery medium, mix well, and transfer to a 1.5 mL centrifuge tube. Incubate at 30 °C for 3 h. Spread 100 μL of the culture onto a YPD+Z antibiotic plate and incubate at 30 °C. Once single colonies appear, spread them onto bleomycin-resistant plates (200-500 μg / mL) for gradient selection. After single colonies of Pichia pastoris have grown, select 10 single colonies for PCR verification, and select positive colonies for induction of expression.

[0052] Pick a single colony and inoculate it into 10 mL of BMGY medium (50 mL shake flask), incubate at 28°C and shake at 200 rpm until OD500 is reached. 600 =6-8 (logarithmic growth phase, approximately 16-18 hours). Centrifuge at 4500 rpm for 5 minutes at room temperature, collect cells, remove supernatant, and resuspend cells in BMMY medium to OD. 600 =1.0, and expression was induced. The induction time was 96 h, with methanol added every 24 h to a final concentration of 1% to continue induction. After reaching the induction time, the cells were centrifuged at 4500 rpm for 5 min at 4℃, and the supernatant was collected for protein gel electrophoresis to detect protein expression in Pichia pastoris. See details for experimental results. Figure 3 .

[0053] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A recombinant keratin K35M, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

2. An expression vector for recombinant keratin K35M, characterized in that: The expression vector is formed by integrating the nucleotide fragment corresponding to recombinant keratin K35M into the multiple cloning site of an empty vector.

3. The expression vector for recombinant keratin K35M according to claim 2, characterized in that: The nucleotide fragment corresponding to recombinant keratin K35M is a nucleotide fragment optimized according to the codon preference of the protein expression host.

4. The expression vector for recombinant keratin K35M according to claim 3, characterized in that: The protein expression host is Escherichia coli or yeast.

5. The expression vector for recombinant keratin K35M according to claim 4, characterized in that: The empty carrier is pET-28a(+) or pPICZαA.

6. A Pichia pastoris expression system for recombinant keratin K35M, characterized in that: This includes Pichia pastoris transformed with an expression vector containing recombinant keratin K35M.

7. The Pichia pastoris expression system for recombinant keratin K35M according to claim 6, characterized in that: It is prepared by the following method: a nucleotide fragment with a sequence such as SEQ ID NO. 3 is integrated into the multiple cloning site of pPICZαA to obtain an expression vector; the expression vector is linearized and then electroporated into competent cells of Pichia pastoris, and then subjected to resistance screening and PCR verification to obtain the Pichia pastoris expression system.

8. The method for preparing recombinant keratin K35M using a Pichia pastoris expression system according to claim 7, characterized in that: The Pichia pastoris expression system was inoculated and cultured in shake flasks until OD500. 600 The value was 6-8; then methanol was used to induce the expression of recombinant keratin K35M; after the expression was induced, the liquid part was separated by solid-liquid separation, and the liquid part was enriched with recombinant keratin K35M.

9. The application of the recombinant keratin K35M according to claim 1 in the preparation of calcium ion chelating agents.

10. The application of the recombinant keratin K35M according to claim 1 in the preparation of oral repair formulations.

Images