Efficient expression and crystallization method of Chlamydomonas reinhardtii HydA2 protein
By modifying the pET-32a vector and combining it with the E. coli heterologous expression system, the problems of low expression efficiency and oxygen sensitivity of the HydA2 protein were solved, achieving efficient soluble expression and crystallization, which supports subsequent structural analysis and improved hydrogen production efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ANHUI UNIV
- Filing Date
- 2026-02-11
- Publication Date
- 2026-05-08
AI Technical Summary
In existing technologies, the HydA2 protein of Chlamydomonas reinhardtii suffers from problems such as low expression efficiency, misfolding, and loss of activity, resulting in severe oxygen sensitivity and affecting the hydrogen production efficiency of green algae.
By modifying the pET-32a vector using homologous recombination technology, the pET-32a-TEV expression vector was constructed. Combined with the E. coli heterologous expression system and purification technology, efficient soluble expression of HydA2 protein and optimization of crystallization conditions were achieved.
We successfully achieved efficient soluble expression and crystallization of the HydA2 protein, ensuring the integrity of the protein's active site structure and providing an experimental basis for subsequent structural analysis and improvement of hydrogen production efficiency.
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Figure CN121991906A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of genetic engineering and protein engineering technology, and in particular to a method for efficient expression and crystallization of the Chlamydomonas reinhardtii HydA2 protein. Background Technology
[0002] Green algae can extract protons and electrons from water through a sunlight-driven biological process, converting chemical energy into environmentally friendly hydrogen (H2). The core of this process is the [FeFe]-hydrogenase expressed by green algae, a highly active biocatalyst that catalyzes the reversible reduction of protons to molecular hydrogen and is linked to the photosynthetic electron transport chain via ferroredoxin PETF. [FeFe]-hydrogenase has a dual function in organisms: oxidizing hydrogen to provide energy and capturing and releasing electrons. Due to its high catalytic efficiency, its biotechnology application in producing H2 from renewable resources represents a potential sustainable solution to address climate change.
[0003] To date, many mechanisms of hydrogen metabolism in algae remain unclear. Most characterized Chlamydomonas hydrogenases consist only of an active site domain (H cluster), without additional domains or iron-sulfur (FeS) clusters. This active site consists of a "standard" [4Fe4S]-cluster (4FeH), which is linked by cysteine residues to a unique [2Fe2S]-cluster (2FeH). This cluster coordinates several non-proteogen ligands (two CN, three CO, and one bridging aziridine disulfide adt). In addition to coordinating the so-called H cluster with the four cysteine ligands, the protein environment surrounding this active site (secondary ligand sphere) influences ligand geometry, redox state, and the stability of the H cluster through various electrostatic contacts and H-bond networks.
[0004] Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii As a typical model organism of green algae, *Chlamydomonas reinhardtii* encodes two [FeFe]-hydrogenases (HydA1 and HydA2) that can independently catalyze the reduction of protons to H2. HydA1 is the main functional protein for H2 production in algae, connecting to the photosynthetic electron transport pathway via phytoredoxin PETF. HydA2, however, accounts for approximately 25% of hydrogen production in algal metabolism, but its specific mechanism of action remains unclear. *Chlamydomonas reinhardtii*'s HydA2, as a double-ferrous hydrogenase, exhibits superior catalytic activity compared to [NiFe]-hydrogenases, making it an important research target in the field of hydrogen production catalysts. However, *Chlamydomonas reinhardtii* hydrogenases are extremely sensitive to oxygen, resulting in very low hydrogen production efficiency, which has become a core bottleneck restricting the practical application of *Chlamydomonas reinhardtii* hydrogen production technology.
[0005] Green algae possess various ferredoxin subtypes, which can regulate electron flow under different environments through specific interactions with different metabolic redox chaperones. Furthermore, the types of ferredoxins bound to different hydrogenases vary. Currently, preparing efficiently expressed *Chlamydomonas reinhardtii* HydA2 and circumventing its inhibition of hydrogenase activity by regulating oxygen concentration are key research directions for improving the photosynthetic hydrogen production efficiency of green algae. Simultaneously, the spatial structure of *Chlamydomonas reinhardtii* HydA2 has not yet been resolved. Determining this protein structure can provide support for research on its physiological and biochemical pathways, thereby helping to find methods to improve its oxygen tolerance and ultimately enhance hydrogen production efficiency. Therefore, achieving efficient active expression of *Chlamydomonas reinhardtii* HydA2 protein and exploring its crystallization conditions are important research directions for improving the photosynthetic hydrogen production efficiency of green algae.
[0006] pET-32a is a commonly used prokaryotic expression vector. Its built-in thioredoxin tag (Trx) helps eukaryotic proteins fold correctly into their native conformation in *E. coli*, while also possessing antioxidant and disulfide bond reduction regulation functions, protecting oxygen-sensitive proteins from oxidative damage and meeting the heterologous expression requirements of the HydA2 protein. Homologous recombination technology enables precise assembly of DNA fragments, providing technical support for the targeted modification of the vector. Based on this, modifying the pET-32a vector using homologous recombination technology to achieve efficient expression and crystallization of the *Chlamydomonas reinhardtii* HydA2 protein is of great significance for research on hydrogen production in green algae.
[0007] In summary, there is an urgent need to develop a method for efficient and active expression of the HydA2 protein from Chlamydomonas reinhardtii, while simultaneously exploring its crystallization conditions. This would address issues such as low expression efficiency, folding errors, and loss of activity caused by oxygen sensitivity in the HydA2 protein, elucidate its protein structure, and provide an experimental basis for improving its oxygen tolerance and hydrogen production efficiency. Summary of the Invention
[0008] The technical problem to be solved by this invention is to provide a method for efficient expression and crystallization of the HydA2 protein from Chlamydomonas reinhardtii. This method involves modifying the pET-32a vector using homologous recombination technology to construct the pET-32a-TEV expression vector, combined with a heterologous expression system in E. coli and purification, to achieve efficient soluble expression, activity retention, and optimized crystallization conditions for the HydA2 protein from Chlamydomonas reinhardtii. This solves the problems of oxygen sensitivity leading to expression folding errors, activity loss, and low expression efficiency in existing technologies for the HydA2 protein. Simultaneously, it obtains effective conditions suitable for the crystallization of the HydA2 protein, laying an experimental foundation for the structural analysis of this protein and research on improving hydrogen production efficiency in green algae.
[0009] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A highly efficient method for expressing the HydA2 protein from Chlamydomonas reinhardtii includes the following steps: (1) Construction of pET-32a-TEV-HydA2 recombinant expression vector Using the *Chlamydomonas reinhardtii* HydA2 gene with the nucleotide sequence shown in SEQ ID NO.1 as a template, a target gene fragment with BamHI / SalI double restriction sites was prepared. This gene fragment was then ligated to the pET-32a vector after BamHI / SalI double restriction to obtain the pET-32a-HydA2 recombinant plasmid. A TEV restriction site was then added downstream of the BamHI restriction site in the recombinant plasmid using homologous recombination technology. After identification and sequencing, the pET-32a-TEV-HydA2 recombinant expression vector was obtained. (2) Obtaining positive recombinant strains The pET-32a-TEV-HydA2 recombinant expression vector was transformed into competent Escherichia coli cells, and single colonies were picked after culture for identification to obtain positive recombinant strains. (3) Inducible expression of HydA2 protein in Chlamydomonas reinhardtii After the positive recombinant strain was expanded and cultured, IPTG was added for induction culture. After the culture was completed, the bacterial cells were collected by centrifugation. (4) Purification of HydA2 protein from Chlamydomonas reinhardtii The collected bacterial cells were lysed, broken, and centrifuged, and the supernatant was collected. The supernatant was then purified by Ni-NTA affinity chromatography and a chromatography system to obtain the target Chlamydomonas reinhardtii HydA2 protein.
[0010] As one of the preferred embodiments of the present invention, in step (1), the method for preparing the target gene fragment is as follows: using the artificially synthesized Chlamydomonas reinhardtii HydA2 gene as a template, PCR amplification is performed using primers F1 and R1; the nucleotide sequences of primers F1 and R1 are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.
[0011] As one of the preferred embodiments of the present invention, in step (1), the nucleotide sequence of the TEV restriction site is shown in SEQ ID NO.4; the homologous recombination technology uses the double-stranded fragment formed by the reverse complementary primers F2 and R2 as the target fragment, and the target fragment is recombinated with the pET-32a-HydA2 recombinant plasmid linearized by BamHI single enzyme digestion; the nucleotide sequences of the primers F2 and R2 are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.
[0012] As one of the preferred embodiments of the present invention, in step (2), the competent Escherichia coli cells are DH5α competent cells; the identification is at least one of PCR identification and double enzyme digestion identification.
[0013] As one of the preferred embodiments of the present invention, in step (3), the positive recombinant strain is cultured to the OD value of the bacterial culture. 600 When the concentration reaches 0.6~0.8, add IPTG. The final concentration of IPTG is 0.4mM. Induce culture at low temperature of 16℃ for 12~16h.
[0014] As one of the preferred embodiments of the present invention, in step (4), the lysis buffer used is a mixture of 20 mM Tris-HCl and 200 mM NaCl, pH 7.5; the disruption is performed by ultrasonic disruption on ice; and the chromatography system is a Superdex 200 chromatography system.
[0015] As one of the preferred embodiments of the present invention, in step (4), Ni-NTA affinity chromatography is performed by imidazole gradient elution, and the elution buffer is a mixture containing 20mM Tris-HCl, 150mM NaCl and 20~500mM imidazole.
[0016] A method for crystallizing Chlamydomonas reinhardtii HydA2 protein involves using the Chlamydomonas reinhardtii HydA2 protein prepared by the above method as raw material, and employing a sitting drop method to screen and optimize the crystals to obtain Chlamydomonas reinhardtii HydA2 protein crystals.
[0017] As one of the preferred methods of the present invention, the Chlamydomonas reinhardtii HydA2 protein is first concentrated to 6 mg / mL, and then subjected to centrifugation and filtration before crystal screening; the sitting drop method uses a 48-well crystal plate, with 200 μL of pooling solution added to the well, and the protein sample and pooling solution mixed in a 1:1 volume ratio in the wells, sealed and cultured, and the crystal growth is observed.
[0018] As one of the preferred embodiments of the present invention, the crystal growth conditions are as follows: using 0.2M lithium sulfate, 0.1MBIS-TRIS, and 25% (w / v) polyethylene glycol 3350 as the pool solution, with a pool solution pH of 6.5, and culturing at 18°C for 7 days.
[0019] The advantages of this invention compared to the prior art are: (1) This invention constructs the HydA2 gene in a pET-32a vector with a thioredoxin tag (Trx) and introduces it into the TEV restriction site via homologous recombination technology. The Trx tag not only helps eukaryotic proteins fold correctly in Escherichia coli to form their native conformation, but also protects HydA2 from oxidative damage by virtue of its antioxidant and disulfide bond reduction regulation properties. At the same time, the TEV enzyme can precisely remove the tag, completely solving the problems of folding errors, loss of activity, and low expression levels when expressing this oxygen-sensitive protein using traditional vectors, and greatly improving the soluble expression efficiency and activity of the target protein. Furthermore, the recombinant protein expressed has characteristic absorption peaks at 318 nm and 420 nm, proving that its active center has a correctly conformed Fe-S cluster structure.
[0020] (2) Based on the extreme sensitivity of HydA2 to oxygen, this invention combines low-temperature induction culture with the antioxidant properties of the Trx tag to construct a heterologous expression scheme for oxygen-sensitive proteins in Escherichia coli. This invention successfully achieves efficient heterologous expression of HydA2 protein from the eukaryotic Chlamydomonas reinhardtii in prokaryotic Escherichia coli, providing a reference for the efficient heterologous expression of similar oxygen-sensitive proteins.
[0021] (3) Compared with traditional enzyme digestion-ligation technology, the homologous recombination technology used in this invention relies on recombinase-mediated sequence homology to achieve precise assembly of DNA fragments. By introducing specific homologous arms at the primer end and precisely aligning the reading frame, it avoids the limitations of base mismatch and enzyme digestion sites. It also has excellent multi-fragment one-step assembly capability, which greatly improves the flexibility and success rate of complex vector construction. Based on this technology, the modification of the pET-32a vector is simple, precise and efficient. The modified pET-32a-TEV vector has the dual advantages of tag-assisted expression and enzyme digestion to remove the tag, providing a practical and feasible technical solution for the targeted modification of related vectors.
[0022] (4) This invention is the first to systematically screen and optimize the crystallization conditions of Chlamydomonas reinhardtii HydA2 protein, determine the optimal parameters for protein crystallization, and successfully obtain the target protein crystal, laying an experimental foundation for subsequent analysis of the spatial structure of HydA2 protein, exploration of its catalytic mechanism and oxygen tolerance-related sites.
[0023] (5) This invention establishes a complete and efficient process for HydA2 protein cloning, induced expression, Ni-NTA affinity chromatography combined with Superdex 200 fine purification. The operation is standardized and the purification effect is good. It can realize the large-scale preparation of active HydA2 protein, which not only meets the sample requirements for subsequent protein structure research and hydrogen production-related application research, but also provides a material basis for exploring the physiological and biochemical pathways of HydA2 and finding its oxygen tolerance modification method. It provides key technical support for solving the industry bottleneck problem of low hydrogen production efficiency of Chlamydomonas from the protein level, and helps to improve the photosynthetic hydrogen production efficiency of green algae by modifying HydA2. Attached Figure Description
[0024] Figure 1 This is an SDS-PAGE electrophoresis image of the experimental expression of HydA2 protein in Chlamydomonas reinhardtii in Example 1 of this invention; Figure 2 This is an SDS-PAGE electrophoresis image of the Ni-NTA affinity chromatography elution fraction of Chlamydomonas reinhardtii HydA2 protein in Example 1 of this invention (in the image, FT refers to the flow-through solution). Figure 3 This is a chromatogram of the Chlamydomonas reinhardtii HydA2 protein in Example 1 of this invention, after fine purification by FPLC using a Superdex 200 chromatography system; Figure 4 This is an SDS-PAGE electrophoresis image of Chlamydomonas reinhardtii HydA2 protein after FPLC fine purification in Example 1 of this invention; Figure 5 This is an SDS-PAGE electrophoresis image of the Chlamydomonas reinhardtii HydA2 protein expression assay in Example 2 of this invention; Figure 6 This is an SDS-PAGE electrophoresis image of the Ni-NTA affinity chromatography elution fraction of Chlamydomonas reinhardtii HydA2 protein in Example 2 of this invention (in the image, FT refers to the flow-through solution). Figure 7 This is a chromatogram of the Chlamydomonas reinhardtii HydA2 protein finely purified by FPLC using a Superdex 200 chromatography system in Example 2 of this invention; Figure 8 This is an SDS-PAGE electrophoresis image of Chlamydomonas reinhardtii HydA2 protein after FPLC fine purification in Example 2 of this invention; Figure 9 This is an SDS-PAGE electrophoresis image of HydA2 protein purified with purification buffer containing 2 mM DTT in Example 2 of this invention. Figure 10This is a full-wavelength scan spectrum of Chlamydomonas reinhardtii HydA2 protein and thioredoxin purified in Example 1 of this invention (Figure A shows the full-wavelength scan result of pET-32a-TEV-HydA2, and Figure B shows the full-wavelength scan result of the thioredoxin tag Trx). Figure 11 This is a crystal image of the Chlamydomonas reinhardtii HydA2 protein obtained in Example 3 of this invention. Detailed Implementation
[0025] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the culture media, vectors, plasmids, and reagents used in the following embodiments and comparative examples are conventional culture media, vectors, plasmids, and reagents in this technical field; and unless otherwise specified, the methods and equipment used in the following embodiments and comparative examples are conventional methods and equipment in this technical field.
[0026] Example 1: Expression of HydA2 protein in Chlamydomonas reinhardtii: 1. Construction of pET-32a-HydA2 recombinant expression vector Using the artificially synthesized Chlamydomonas reinhardtii HydA2 gene (SEQ ID NO.1) as a template, PCR amplification was performed using primers F1 (SEQ ID NO.2) and R1 (SEQ ID NO.3) to obtain the HydA2 gene fragment with BamHI / SalI double restriction sites. This gene fragment was then double-digested with the pET-32a vector using BamHI / SalI, followed by ligation using T4 DNA rapid ligase at 25°C. The ligation system was transformed into DH5α competent cells, plated, and incubated overnight at 37°C. The next day, single clones were picked and incubated overnight at 37°C in LB medium at 220 rpm. Recombinant plasmids were then extracted, confirmed by double restriction enzyme digestion, and sent to the company for sequencing to obtain the pET-32a-HydA2 recombinant plasmid.
[0027] 2. Construction of the pET-32a-TEV-HydA2 recombinant expression vector Using homologous recombination technology, a TEV restriction site (SEQ ID NO.4) was introduced downstream of the BamHI restriction site in the pET-32a-HydA2 recombinant plasmid. The specific operation is as follows: (1) The pET-32a-HydA2 recombinant plasmid was digested with BamHI to make it a linearized vector for later use; (2) Using reverse complementary primer F2 (SEQ ID NO.5) and primer R2 (SEQ ID NO.6), 5 μL of each of the upstream and downstream primers were added to 40 μL of ddH2O to prepare the reaction system. The system was heated in a 95℃ water bath for 10 min and then slowly cooled to room temperature so that primers F2 and R2 could bind complementaryly to form a double-stranded target fragment with TEV restriction sites. (3) Prepare a recombination system with a total volume of 10 μL, specifically consisting of: 0.8 μL of linearized pET-32a-HydA2 vector, 3.2 μL of double-stranded target fragment, and 5 μL of 2×Basic Assembly Mix. Place the system at 50 °C for 10 min to complete homologous recombination. (4) The above recombinant reaction solution was transformed. Several single colonies were picked from the transformation plate and inoculated into 4 mL of LB liquid medium containing the corresponding antibiotic. The medium was cultured at 37°C with shaking for 14 h. The plasmid DNA of the recombinant clone was extracted and subjected to PCR, double enzyme digestion detection and sequencing verification. After identification and correct sequencing, the pET-32a-TEV-HydA2 recombinant expression vector was obtained.
[0028] 3. Obtaining positive recombinant strains The pET-32a-TEV-HydA2 recombinant expression vector was transformed into Escherichia coli DH5α competent cells. After transformation, several single colonies were picked from the transformation plate and inoculated into 4 mL of LB liquid medium containing ampicillin. The cells were then cultured at 37°C with shaking for 14 h. Subsequently, the plasmid DNA of the recombinant clone was extracted and subjected to PCR, double enzyme digestion detection, and sequencing verification. After identification and confirmation of correct sequencing, a positive recombinant strain was obtained.
[0029] 4. Experimental expression of HydA2 protein from Chlamydomonas reinhardtii The positive recombinant strain was inoculated into 4 mL of LB liquid medium containing ampicillin and cultured at 37°C with shaking until the bacterial culture reached OD. 600 When the concentration reached approximately 0.6, a portion of the bacterial culture was used as a pre-induction control. IPTG was added to the remaining bacterial culture to a final concentration of 0.4 mM, and expression was induced for 4 hours. 1 mL of the bacterial culture was then collected as a post-induction sample. The bacterial cells were collected by centrifugation, resuspended in an appropriate amount of 2×SDS loading buffer, boiled in a water bath for 10 min, centrifuged again, and the supernatant was used for SDS-PAGE to detect the expression of recombinant HydA2 protein. The results are as follows: Figure 1 As shown, the target protein pET-32a-TEV-HydA2 has a molecular weight of 68.02 kDa.
[0030] 5. Increased expression of HydA2 protein in Chlamydomonas reinhardtii The recombinant clonal strain expressing the HydA2 protein was inoculated into 4 mL of LB broth containing ampicillin and incubated overnight at 37°C. The next day, the inoculum was expanded to 1 L of LB broth containing ampicillin at a 1:100 dilution and cultured at 37°C with shaking until OD (dose retardation). 600 When the bacterial cell growth rate reaches 0.6-0.8, add IPTG to a final concentration of 0.4 mM, and incubate at 16°C for 12 h. Then, collect the bacterial cells by centrifugation. After lysing and centrifuging on ice, collect the supernatant and precipitate separately, add an appropriate amount of 2×SDS loading buffer, and perform SDS-PAGE to detect whether the protein is soluble and expressed.
[0031] 6. Expression and purification of HydA2 protein from Chlamydomonas reinhardtii (1) The collected bacterial pellet was resuspended in lysis buffer (20mM Tris-HCl pH 7.5, 200mM NaCl) and placed on ice for sonication to disrupt the cells for two rounds, each round for 30-40 min; after disruption, the disruption solution was centrifuged at 4℃ and 14000rpm for 40 min, and the supernatant was collected, which is the crude enzyme solution containing the target protein.
[0032] (2) The crude enzyme solution was slowly loaded onto a Ni-NTA chromatography column equilibrated with equilibration buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl). After loading, the protein was eluted sequentially using gradient elution buffers: a) 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM imidazole; b) 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 200 mM imidazole; c) elution buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 500 mM imidazole. The eluted fractions were collected and detected by 12% SDS-PAGE electrophoresis. Figure 2 The imidazole concentration of the elution buffer containing the target protein was determined; the Bradford protein quantification kit was used to detect the content of the target protein in the elution buffer containing the target protein.
[0033] (3) The fraction containing the target protein obtained by Ni-NTA affinity chromatography was further purified by FPLC using a Superdex 200 chromatography system. Figure 3 To remove contaminating proteins and residual imidazole; the eluted fraction was collected and analyzed by 12% SDS-PAGE electrophoresis. Figure 4 We obtained purified, highly soluble, and active Chlamydomonas reinhardtii HydA2 protein.
[0034] Example 2: Using pET 28a vector expression of Chlamydomonas reinhardtii HydA2 protein: 1. Construction of pET-32a-HydA2 recombinant expression vector Using the artificially synthesized Chlamydomonas reinhardtii HydA2 gene (SEQ ID NO.1) as a template, PCR amplification was performed using primers adapted to Nde I and Xho I restriction sites synthesized by a commissioned biotechnology company to obtain a HydA2 gene fragment with Nde I / Xho I double restriction sites. The primers used for PCR amplification and subsequent experiments were all directly synthesized by the commissioned biotechnology company. This gene fragment was double-digested with the pET-28a vector using Nde I / Xho I, and then ligated using T4 DNA rapid ligase at 25°C. The ligation system was transformed into DH5α competent cells, plated, and incubated overnight at 37°C. The next day, single clones were picked and incubated overnight at 37°C in LB medium at 220 rpm. Recombinant plasmids were then extracted, confirmed by double enzyme digestion, and sent to the company for sequencing to obtain the pET-28a-HydA2 recombinant plasmid.
[0035] 2. Obtaining positive recombinant strains The pET-28a-HydA2 recombinant expression vector was transformed into Escherichia coli DH5α competent cells. After transformation, several single colonies were picked from the transformation plate and inoculated into 4 mL of LB liquid medium containing kanamycin (the pET-28a vector carries the kanamycin resistance gene). The cells were cultured at 37°C with shaking for 14 h. Subsequently, the plasmid DNA of the recombinant clone was extracted and subjected to PCR, double enzyme digestion detection, and sequencing verification. After identification and confirmation of correct sequencing, a positive recombinant strain was obtained.
[0036] 3. Trial and expanded expression of HydA2 protein from Chlamydomonas reinhardtii The operating steps are completely consistent with steps 4 (trial expression) and 5 (expanded expression) in Example 1: During the trial expression, the positive recombinant strain was inoculated into 4 mL of LB liquid medium containing kanamycin and cultured at 37°C with shaking until OD. 600 When the expression level reached approximately 0.6, IPTG was added to a final concentration of 0.4 mM for 4 hours of induction. SDS-PAGE was then used to detect expression levels. The results are as follows: Figure 5 As shown, the target protein pET-28a(+)-His6-CrHydA2 has a molecular weight of 47.02 kDa.
[0037] For expanded expression, inoculate at a rate of 1:100 to 1 L of LB liquid medium containing kanamycin and incubate at 37°C until OD500 is reached. 600 The concentration was 0.6~0.8, IPTG was added to a final concentration of 0.4mM, and the cells were induced at 16℃ for 12h. The cells were collected by centrifugation, and the protein solubility was tested after lysis.
[0038] 4. Expression and purification of HydA2 protein from Chlamydomonas reinhardtii The operation steps are completely consistent with step 6 (expression and purification) in Example 1, as follows: (1) The collected bacterial pellet was resuspended in lysis buffer (20mM Tris-HCl pH 7.5, 200mM NaCl) and placed on ice for sonication to disrupt the cells for two rounds, each round for 30-40 min; after disruption, the disruption solution was centrifuged at 4℃ and 14000rpm for 40 min, and the supernatant was collected, which is the crude enzyme solution containing the target protein.
[0039] (2) The crude enzyme solution was slowly loaded onto a Ni-NTA chromatography column equilibrated with equilibration buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl). After loading, the protein was eluted sequentially using gradient elution buffers: a) 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM imidazole; b) 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 200 mM imidazole; c) elution buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 500 mM imidazole. The eluted fractions were collected and detected by 12% SDS-PAGE electrophoresis. Figure 6 The imidazole concentration of the elution buffer containing the target protein was determined; the Bradford protein quantification kit was used to detect the content of the target protein in the elution buffer containing the target protein.
[0040] (3) The fraction containing the target protein obtained by Ni-NTA affinity chromatography was further purified by FPLC using a Superdex 200 chromatography system. Figure 7 To remove contaminating proteins and residual imidazole; the eluted fraction was collected and analyzed by 12% SDS-PAGE electrophoresis. Figure 8 ).
[0041] The above results show that the HydA2 protein expressed on the pET-28a vector is a dimer, but many bands appear near the target protein. Furthermore, the addition of 2 mM DTT to the protein purification buffer, even when using a protein purification buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 2 mM DTT, did not improve this phenomenon. Figure 9 The results indicate that pET-28a-HydA2 protein folds incorrectly when heterologously expressed in E. coli. This may be because E. coli is cultured under aerobic conditions, while HydA2 is extremely sensitive to oxygen. Contact with oxygen causes it to lose its activity and its spatial structure to change.
[0042] Based on the results of Example 1 and Comparative Example 1, it can be seen that the recombinant protein expressed by pET-32a-TEV-HydA2 in this invention effectively improves the phenomenon of incorrect protein folding. At the same time, it was found that under the same culture conditions, the peak value of FPLC purification of pET-32a-TEV-HydA2 was much higher than that of pET-28a-HydA2, indicating that HydA2 can be expressed more efficiently on the pET-32a vector.
[0043] Furthermore, since HydA2 is a diferroic hydrogenase with a unique active site—the H cluster—containing a [2Fe-2S] center (a [4Fe-4S] center bridged to the catalytic site (H cluster), a correctly folded diferroic hydrogenase will exhibit a specific absorption peak. Therefore, a full-wavelength scan was performed on the obtained protein sample and the excised thioredoxin. The results... Figure 10 As shown.
[0044] The scanning results revealed that the pET-32a-TEV-HydA2 recombinant protein, in addition to a protein absorption peak at 280 nm, also exhibited specific absorption peaks at 318 nm and 420 nm, indicating that the active site of this protein has a correctly conformed Fe-S cluster structure.
[0045] In summary, this invention successfully achieved highly efficient and soluble expression of the Chlamydomonas reinhardtii Hydrogenase HydA2 protein in Escherichia coli by constructing the pET-32a-TEV-HydA2 recombinant expression vector and combining it with the thioredoxin tag's folding-assisted and antioxidant properties. The obtained protein was correctly folded and had an intact Fe-S cluster structure at the active site, which can be used for subsequent crystal screening and structural analysis. In contrast, the traditional pET-28a vector expression system suffers from problems such as protein folding errors, low solubility, and lack of activity, failing to meet research requirements. This fully demonstrates the significant technical advantages of this invention in the field of heterologous expression of oxygen-sensitive proteins.
[0046] Example 3: Crystallization of HydA2 protein from Chlamydomonas reinhardtii: The purified HydA2 protein solution obtained by FPLC (Example 1) was collected, centrifuged, filtered, and concentrated. The protein concentration was determined using a one-drop method. The purified HydA2 protein was screened for crystals using the sitting drop method. 200 μL of pooling solution was added to each well of a 48-well crystallizer, followed by 1 μL of sample and then 1 μL of pooling solution. The wells were sealed with special crystallizer tape to ensure a closed system. The wells were labeled and placed on shelves in a cold storage at 4°C or 16°C. Crystal growth was observed multiple times after crystallization, one day, three days, and one week.
[0047] The initial crystal screening conditions were: Hampton and QIAGEN crystallography kits. The Hampton kit screened for the following conditions: Index, SaltRx 1, SaltRx 2, PEGRx 1, PEGRx 2, CrystalScreen, Crystal Screen 2. The QIAGEN kit screened for the following conditions: NeXtal Tubes AmSO4 Suite, NeXtal Tubes Classics Suite, NeXtal Tubes Classics II Suite, NeXtal Tubes Classics L Suite, NeXtal Tubes PEGs Suite, NeXtal Tubes PEGs II Suite, NeXtal Tubes Cryos Suite, and NeXtal Tubes Nucleix Suite.
[0048] Preliminary crystal screening revealed crystal formation at a protein concentration of 6 mg / mL after 7 days of incubation at 18°C in 0.2M lithium sulfate, 0.1M BIS-TRIS, and 25% (w / v) polyethylene glycol 3350. Figure 11 The discovery of this crystal laid an important experimental foundation for subsequent analysis of the spatial structure of the Chlamydomonas reinhardtii HydA2 protein, elucidation of its catalytic mechanism and oxygen-sensitivity molecular mechanism, and targeted modification to obtain oxygen-tolerant, highly active hydrogenase.
[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for efficient expression of HydA2 protein from Chlamydomonas reinhardtii, characterized in that, Includes the following steps: (1) Construction of pET-32a-TEV-HydA2 recombinant expression vector Using the *Chlamydomonas reinhardtii* HydA2 gene with the nucleotide sequence shown in SEQ ID NO.1 as a template, a target gene fragment with BamHI / SalI double restriction sites was prepared. This gene fragment was then ligated to the pET-32a vector after BamHI / SalI double restriction to obtain the pET-32a-HydA2 recombinant plasmid. A TEV restriction site was then added downstream of the BamHI restriction site in the recombinant plasmid using homologous recombination technology. After identification and sequencing, the pET-32a-TEV-HydA2 recombinant expression vector was obtained. (2) Obtaining positive recombinant strains The pET-32a-TEV-HydA2 recombinant expression vector was transformed into competent Escherichia coli cells, and single colonies were picked after culture for identification to obtain positive recombinant strains. (3) Inducible expression of HydA2 protein in Chlamydomonas reinhardtii After the positive recombinant strain was expanded and cultured, IPTG was added for induction culture. After the culture was completed, the bacterial cells were collected by centrifugation. (4) Purification of HydA2 protein from Chlamydomonas reinhardtii The collected bacterial cells were lysed, broken, and centrifuged, and the supernatant was collected. The supernatant was then purified by Ni-NTA affinity chromatography and a chromatography system to obtain the target Chlamydomonas reinhardtii HydA2 protein.
2. The expression method according to claim 1, characterized in that, In step (1), the preparation method of the target gene fragment is as follows: using the artificially synthesized Chlamydomonas reinhardtii HydA2 gene as a template, PCR amplification is performed using primers F1 and R1; the nucleotide sequences of primers F1 and R1 are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.
3. The expression method according to claim 1, characterized in that, In step (1), the nucleotide sequence of the TEV restriction site is shown in SEQ ID NO.4; the homologous recombination technology uses the double-stranded fragment formed by the reverse complementary primers F2 and R2 as the target fragment, and performs a recombination reaction with the pET-32a-HydA2 recombinant plasmid linearized by BamHI single enzyme digestion; the nucleotide sequences of the primers F2 and R2 are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.
4. The expression method according to claim 1, characterized in that, In step (2), the competent Escherichia coli cells are DH5α competent cells; the identification is at least one of PCR identification and double enzyme digestion identification.
5. The expression method according to claim 1, characterized in that, In step (3), the positive recombinant strain is cultured to the OD value of the bacterial culture. 600 When the concentration reaches 0.6~0.8, add IPTG to a final concentration of 0.4mM, and induce culture at 16℃ for 12~16h.
6. The expression method according to claim 1, characterized in that, In step (4), the lysis buffer used is a mixture of 20mM Tris-HCl and 200mM NaCl, pH 7.5; the disruption is performed by sonication on ice; and the chromatography system is a Superdex 200 chromatography system.
7. The expression method according to claim 1, characterized in that, In step (4), Ni-NTA affinity chromatography uses imidazole gradient elution, and the elution buffer is a mixture containing 20mM Tris-HCl, 150mM NaCl and 20~500mM imidazole.
8. A method for crystallizing Chlamydomonas reinhardtii HydA2 protein, characterized in that, Using the Chlamydomonas reinhardtii HydA2 protein prepared by any one of claims 1 to 7 as raw material, the crystals were screened and optimized by the sitting drop method to obtain Chlamydomonas reinhardtii HydA2 protein crystals.
9. The crystallization method according to claim 8, characterized in that, The Chlamydomonas reinhardtii HydA2 protein was first concentrated to 6 mg / mL, and then subjected to centrifugation and filtration before crystal screening. The drop method was performed using a 48-well crystal plate. 200 μL of pooling solution was added to the well, and the protein sample and pooling solution were mixed in a 1:1 volume ratio. The plate was then sealed and cultured, and the crystal growth was observed.
10. The crystallization method according to claim 8, characterized in that, The crystal growth conditions were as follows: using 0.2M lithium sulfate, 0.1M BIS-TRIS, and 25% (w / v) polyethylene glycol 3350 as the pool solution, with a pool solution pH of 6.5, and incubating at 18°C for 7 days.