A Rubisco protein extraction and analysis method

Abstract

This invention discloses a method for extracting and analyzing Rubisco protein, comprising the following steps: Step 1: providing a raw material extract containing the Rubisco protein to be extracted; Step 2: adding polyethylene glycol to the raw material extract to a final concentration of 0.01-0.05 g / mL, followed by centrifugation and collection of the supernatant; Step 3: adding more polyethylene glycol to the supernatant to a final concentration of 0.08-0.15 g / mL, followed by centrifugation to obtain Rubisco protein precipitate. This invention achieves controllable separation of different proteins based on their solubility by precisely controlling the PEG concentration range, enabling efficient enrichment of the target Rubisco protein while removing most contaminating proteins. Compared to the traditional ammonium sulfate precipitation method, this method offers milder conditions, higher selectivity, and effectively reduces the risk of protein denaturation and activity loss.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for extracting and analyzing Rubisco protein. Background Technology

[0002] Ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) is a key enzyme in photosynthesis that catalyzes CO2 fixation. It is widely found in higher plants, algae, and some microorganisms, and is one of the most abundant proteins on Earth. Rubisco typically assembles into an L8S8 complex composed of eight large subunits (L, approximately 55 kDa) and eight small subunits (S, approximately 15 kDa), with a total molecular weight of approximately 550 kDa. As a crucial enzyme in CO2 fixation during photosynthesis, Rubisco converts inorganic carbon into organic matter. It serves as a central starting point for the Earth's carbon cycle and biosynthesis, playing a pivotal role in carbon cycling, biomanufacturing, and synthetic biology.

[0003] Despite the abundance of Rubisco sources, efficient and high-purity extraction still faces numerous challenges. Firstly, the protein composition of plant leaves is extremely complex, containing not only Rubisco but also a large number of photosystem-related proteins (such as chlorophyll-binding proteins), enzymes (such as Rubisco activase), and impurities such as polyphenols and pigments. These components are prone to co-precipitation with the target protein or non-specific binding during extraction, severely affecting purity. Secondly, Rubisco is sensitive to environmental conditions and is easily affected by oxidation, protease degradation, and conformational changes during extraction, leading to decreased activity or even inactivation. Furthermore, large-scale extraction requires balancing ease of operation with cost control, further increasing the difficulty of process optimization.

[0004] In existing technologies, the extraction of Rubisco mainly relies on methods such as ammonium sulfate fractionation, differential centrifugation, gel filtration chromatography, or ion exchange chromatography. While ammonium sulfate precipitation is simple to operate, it suffers from problems such as high ionic strength, easy induction of protein conformational changes, and cumbersome subsequent desalting steps. Traditional differential centrifugation has limited resolution and is difficult to effectively remove contaminating proteins with similar molecular weights. Chromatographic methods, although capable of achieving high purity, typically suffer from high cost, limited throughput, and complex processes, making them unsuitable for large-scale preparation. Furthermore, some methods fail to effectively control protein aggregation or degradation during purification, resulting in poor homogeneity of the final product, which is detrimental to subsequent structural analysis.

[0005] In structural studies, high-resolution structural resolution of Rubisco relies on protein samples with high purity and homogeneity. However, samples obtained using traditional extraction methods often suffer from residual impurities and a high proportion of aggregates, severely limiting the effectiveness of techniques such as X-ray crystallography and cryo-electron microscopy (Cryo-EM). Therefore, developing a Rubisco extraction method that balances high purity, high activity, and high homogeneity is of great significance for advancing its structural and functional research.

[0006] In summary, existing technologies for Rubisco extraction still suffer from problems such as insufficient purity, loss of activity, complex processes, and difficulty in meeting the needs of structural research. There is an urgent need to develop a novel extraction and purification technology system that is easy to operate, has mild conditions, can be scaled up, and can obtain high-quality protein samples to meet its multiple needs in basic research and application development. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention proposes a method for extracting and analyzing Rubisco proteins.

[0008] A method for extracting Rubisco protein includes the following steps: Step 1: Provide the raw material extract containing the Rubisco protein to be extracted; Step 2: Add polyethylene glycol to the raw material extract to a final concentration of 0.01-0.05 g / mL, then centrifuge and collect the supernatant; Step 3: Add polyethylene glycol to the supernatant to a final concentration of 0.08-0.15 g / mL, and then centrifuge to obtain Rubisco protein precipitate.

[0009] Optionally, the polyethylene glycol is selected from PEG 4000, PEG 6000 or PEG 8000; Preferably, in step two, polyethylene glycol is added to a final concentration of 0.05 g / mL; in step three, polyethylene glycol is further added to the supernatant to a final concentration of 0.10 g / mL. Preferably, the precipitation process in steps two and three involves stirring the mixture evenly at 4°C and then allowing it to stand for 1 hour.

[0010] Optionally, the centrifugation conditions are: temperature: 0–10℃, centrifugal force: 10,000–12,000 g, time: 10 min–40 min, and pH range: 6.5–8.5.

[0011] Optionally, it also includes purification by density gradient centrifugation after resuspending the Rubisco protein precipitate.

[0012] Optionally, the density gradient centrifugation is sucrose density gradient ultracentrifugation, with a sucrose gradient range of 0.2-0.4-0.6 g / mL nonlinear sucrose gradient, a centrifugal force of 100,000-200,000 g, and a centrifugation time of 6-24 hours.

[0013] Optionally, the raw material extract contains: pH buffer solution, Mg 2+ Reducing agents, protease inhibitors, and glycerol; the pH of the raw material extract is 7.0–8.8; Preferably, the pH buffer solution is at least one of the following: Tris-HCl buffer system, PBS buffer system, phosphate buffer system, and HEPES buffer system; Mg 2+ The concentration is 5 mM to 20 mM; The reducing agent includes at least one of tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), and β-mercaptoethanol; the concentration of the reducing agent is 1 mM to 5 mM. The protease inhibitor includes at least one of phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), and leupeptin; the concentration of the protease inhibitor is 1 mM to 5 mM.

[0014] Optionally, the raw material extract is obtained from plant tissue or a microbial or cell culture system expressing Rubisco protein; Preferably, the plant tissue is a leaf from tobacco, spinach, Arabidopsis, rice, or wheat.

[0015] Optionally, the method further includes further fine purification using at least one of gel filtration chromatography, ion exchange chromatography, or affinity chromatography.

[0016] The present invention also proposes a Rubisco protein sample purified by the above method.

[0017] This invention also proposes a method for analyzing Rubisco proteins, comprising: Preparation of Rubisco protein samples; The Rubisco protein sample was then analyzed using cryo-electron microscopy or X-ray crystallography.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention introduces a graded PEG precipitation system into the Rubisco protein extraction process. By precisely controlling the PEG concentration range, it achieves controllable separation of different proteins based on their solubility differences, enabling the efficient enrichment of the target Rubisco protein while removing most contaminating proteins. Compared to the traditional ammonium sulfate precipitation method, this method offers milder conditions, higher selectivity, and effectively reduces the risk of protein denaturation and activity loss.

[0019] This invention combines PEG fractionation precipitation with sucrose density gradient centrifugation to form a two-stage purification system of "coarse separation + fine separation," which not only significantly improves protein purity but also effectively enhances sample homogeneity, providing a high-quality sample source for subsequent structural analysis.

[0020] This invention introduces a complex buffer system containing reducing agents, metal ions, and protease inhibitors during the extraction process, and operates under low temperature conditions, which effectively inhibits oxidative degradation and protein hydrolysis, and significantly improves the activity retention rate and structural stability of Rubisco.

[0021] This invention not only focuses on protein extraction efficiency, but also systematically optimizes process parameters to meet the requirements of cryo-electron microscopy (Cryo-EM) or crystallography for sample purity and homogeneity, achieving an integrated design of "extraction-purification-structure analysis" and improving the success rate of structural studies.

[0022] This invention avoids reliance on high-cost chromatography as the core method, employing a precipitation and centrifugation-based technical route that ensures purity while possessing good scale-up potential, making it suitable for large-scale preparation. High concentration gradients significantly improve the purification yield of Rubisco protein. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This is a flowchart of Embodiment 1 of the present invention; Figure 2 The results are from the SDS-PAGE test in Example 1. Figure 3 The UV-vis absorbance test results are for Example 1; Figure 4 This refers to the mass spectrometry detection result of detection band 1 in Example 1; Figure 5 This refers to the mass spectrometry detection result of detection band 2 in Example 1; Figure 6 This refers to the mass spectrometry detection results of detection band 3 in Example 1; Figure 7 Results of protein morphology determination using transmission electron microscopy; Figure 8 High-resolution three-dimensional structure of Rubisco protein; Figure 9 This is a comparison chart of the results of Example 1, Example 2, and Comparative Example 1; Figure 10 The results are for Comparative Example 2. Figure 11 The results are for Comparative Example 3; Figure 12 The results are for Comparative Example 4. Figure 13 The results are for Comparative Example 5. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0026] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Example 1 like Figure 1As shown, take 200g of fresh tobacco leaves. Add 20g of the following to lysis buffer (50 mM Tris-HCl (pH 7.4) (brand: Dogesce, CAS: 1185-53-1), 1 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP HCl) (brand: Aladdin, CAS: 51805-45-9), 20 mM MgCl2 (brand: Aladdin, CAS: 7786-30-3), 20 mM NaHCO3 (brand: Macklin, CAS: 144-55-8), 1 mM phenylmethanesulfonylfluoride (PMSF) (brand: Macklin, CAS: 329-98-6), 10% glycerol (for protein stabilization) (brand: Macklin, CAS: 56-81-5, total pH 8.8) during lysis. Polyvinylpyrrolidone (PVPP, used for adsorbing polyphenols) (Brand: Sigma-Aldrich, CAS: 9003-39-8). Centrifuge at 12000g for 30 min and collect the supernatant.

[0030] Add 0.1% Triton-X-100 (brand: Aladdin, CAS: 9002-93-1), 0.2M NaCl (brand: Aladdin, CAS: 7647-14-5), and PEG 4000 to the supernatant to a final concentration of 0.05 g / mL. Stir in an ice bath at 4°C for 30 minutes, then let stand for 1 hour. Centrifuge at 12000g for 30 minutes and collect the supernatant. Filter using a 5µm filter membrane to remove any unprecipitated residue.

[0031] Triton-X-100 is used to embed in the lipid bilayer of the cell membrane, interfering with the orderly arrangement of lipid molecules, reducing membrane stability, aiding cell lysis, and releasing the target protein. NaCl is used to maintain osmotic pressure balance, shield the exposed charge on the protein surface, reduce non-specific aggregation of protein molecules due to electrostatic interactions, thereby stabilizing the native conformation of the protein and improving its solubility.

[0032] Add PEG 4000 to the supernatant to a final concentration of 0.10 g / mL, stir in an ice bath at 4°C for 1 h, and let stand for 1 h. Centrifuge at 12000g for 45 min to collect the precipitate, redissolve the precipitate in a small amount of supernatant, and transfer to a 50 mL round-bottom centrifuge tube. Centrifuge again at 12000g for 45 min to collect the precipitate.

[0033] Resuspend the precipitate in 4-6 ml of PBS and mix well for 4 hours (overnight).

[0034] Take 1 mL of protein solution and prepare a 10%–30% (w / v) sucrose density gradient using the Biocomp fully automated density gradient preparation, separation, and collection system (Biocomp, model: 153). Centrifuge at 150,000 g, 4°C, for 16 h using an Optima XE-100 Ultracentrifuge centrifuge (Beckman Coulter, item number: A99836), SW41 Ti horizontal drum rotor (Beckman Coulter, item number: 24U52131), and 13.2 mL Thinwall Polypropylene centrifuge tubes (Beckman Coulter, item number: 331372). Sucrose concentration gradient: 20%–40%–60% (w / v, i.e., 0.2–0.4–0.6 g / mL) nonlinear.

[0035] Rubisco characterization: Based on the high-purity Rubisco protein obtained above, structural characterization was performed, including: (1) Purity analysis: Protein subunit composition was determined using SDS-PAGE; mass spectrometry was used for detection; and UV-vis absorbance was measured. UV-vis absorbance measurement method: Using pure water as a reference, and with the wavelength zeroed at 280 nm, the absorbance values ​​(A) of different samples at 280 nm were measured using the Biocomp fully automated density gradient preparation, separation, and collection system (Brand: Biocomp, Model: 153) under the same conditions. 280 Real-time online detection and mapping of nucleic acids or proteins were performed, and the target components were collected using an FC 203B fraction collector (brand: Gilson, model: FC 203B). Detection results are as follows: Figure 2 , Figure 3 As shown.

[0036] Mass spectrometry detection (sample sent to the Institute of Biophysics, Chinese Academy of Sciences for detection); (detection results of three bands are as follows) Figure 4 , Figure 5 , Figure 6 (As shown).

[0037] like Figure 4 As shown, the detection band corresponds to that originating from fluffy tobacco ( Nicotiana tomentosiformisThe large subunits of ribulose-1,5-bisphosphate carboxylase (Rubisco LSu) have a single isotopic molecular weight of 53,378 Da (approximately 53.4 kDa). The protein identification results are of extremely high confidence: a Mascot score of 184 and an expected value (E-value) as low as 3.3 × 10⁻⁶. - ¹ 4 The sequence coverage reached 62%, and a total of 27 peptides were successfully matched by mass spectrometry data.

[0038] like Figure 5 As shown, the detection band corresponds to that originating from fluffy tobacco ( Nicotiana tomentosiformis The large subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco LSu) has a single isotopic molecular weight of 53,378 Da (approximately 53.4 kDa). The protein identification results are of extremely high confidence: a Mascot score of 247 and an expected value (E-value) as low as 1.6 × 10⁻⁶. -20 The sequence coverage reached 53%, and a total of 27 peptides were successfully matched by mass spectrometry data.

[0039] like Figure 6 As shown, this detection band corresponds to that derived from ordinary tobacco ( Nicotiana tabacum The small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco SSu) has a single isotopic molecular weight of 20,526 Da (approximately 20.5 kDa). The protein identification results have high confidence: a Mascot score of 95 and an expected value (E-value) of 2.5 × 10⁻⁶. -5 The sequence coverage reached 68%, and a total of 12 peptides were successfully matched by mass spectrometry data.

[0040] (2) Morphological analysis: Protein morphology was determined using transmission electron microscopy (TEM); such as Figure 7As shown, the transmission electron microscope (TEM) image reveals a large number of uniformly shaped, nearly spherical granular structures evenly dispersed in the field of view. These particles are approximately 10–15 nm in diameter, with clear boundaries, relatively low central electron density, and a dark halo formed by negative staining. These particles are likely purified Rubisco holoenzyme (L8S8) complexes. Their particle size is highly consistent with the reported size of Rubisco holoenzymes (approximately 10–15 nm). The particles are evenly distributed in the image, and no obvious protein aggregates, degradation fragments, or vacuolar impurities were observed, indicating that the Rubisco sample purified by the sucrose gradient has good homogeneity, intact structure, and excellent dispersion.

[0041] Rubisco structure parsing method The spatial structure of the native Rubisco holoenzyme from *Nicotiana benthamiana* was resolved using cryo-electron microscopy (Cryo-EM) or X-ray crystallography. The high-resolution structure of the holoenzyme was successfully determined. The holoenzyme was naturally extracted from *Nicotiana benthamiana* using sucrose gradient density centrifugation, and purified to obtain a biologically active 16-mer complex (assembled from large and small subunits). Using cryo-electron microscopy (Cryo-EM) single-particle 3D reconstruction technology, the high-resolution 3D structure of this 16-mer complex was resolved for the first time, clarifying its overall conformation, subunit assembly mode, and distribution characteristics of the active center. Figure 8 This provides a core structural template for subsequent research on interaction mechanisms.

[0042] like Figure 8 As shown in the top view (left), the Rubisco holoenzyme exhibits a fourfold symmetrical square ring structure with a distinct central cavity. The large subunits (blue) form the core framework of the enzyme complex, arranged at the four corners and the central region, forming the L8 core framework. The small subunits (pink) are embedded in the gaps between the large subunits, located on either side of the fourfold symmetry axis, playing a role in stabilizing the holoenzyme conformation and regulating enzyme activity. This L8S8 assembly (8 large subunits + 8 small subunits) is a typical characteristic of the higher plant Rubisco.

[0043] As can be seen from the side view (right side, rotated 90°), the holoenzyme complex exhibits a flattened ellipsoidal shape in the vertical direction, with a thickness significantly smaller than its diameter. The upper and lower surfaces have similar structures, further confirming its fourfold symmetrical disc-shaped structure. The large subunits form two layers in the side view, with four in each layer, while the small subunits are distributed in the peripheral region between the two layers, tightly "clamping" the two layers of large subunits together and maintaining the overall stability of the holoenzyme.

[0044] The structural diagram is highly consistent with the aforementioned negative staining results of transmission electron microscopy (spherical particles of about 10–15 nm), confirming at the atomic resolution that the Rubisco obtained by sucrose gradient purification is a complete and correctly assembled functional L8S8 holoenzyme complex, rather than a depolymerized subunit fragment or an incorrectly assembled aggregate.

[0045] Example 2 Compared to Example 1, the PEG concentration in the first precipitation was adjusted to 1%, and the results... Figure 9 .

[0046] Comparative Example 1 Compared to Example 1, the PEG concentration in the first precipitation was adjusted to 8%, and the results... Figure 9 .

[0047] The Native PAGE results showed that both 1% and 5% concentration samples exhibited clear and concentrated blue bands in the Rubisco holoenzyme region from 440 kD to 669 kD. The band positions closely matched the theoretical molecular weight, and the bands were deep and wide, indicating that the intact oligomeric complex was effectively separated and detected, and that the amount of residual Rubisco in the supernatant was relatively abundant. However, the 8% concentration sample showed significantly narrower and lighter bands in the same region, indicating that as the PEG concentration increased, more Rubisco holoenzyme was precipitated, resulting in a significant reduction in the amount of target protein remaining in the supernatant and a decrease in extraction efficiency.

[0048] According to the SDS-PAGE results, both 1% and 5% concentration samples showed clearly distinguishable corresponding bands at approximately 55 kD (large subunit LSU) and approximately 15 kD (small subunit SSu) under denaturing conditions, with moderate band intensity and clean background, indicating that the protein subunit structure was intact and had not been degraded. However, in the 8% concentration sample, under the same dilution factor and loading amount, the target bands at 55 kD and 15 kD were significantly lighter, further confirming that high concentration PEG has a stronger precipitation ability on Rubisco, and the content of soluble LSU and SSu in the supernatant was greatly reduced.

[0049] Therefore, PEG concentrations of 1% and 5% can effectively remove most of the impurities during the first precipitation, while retaining most of the Rubisco in the supernatant, thus achieving the purpose of preliminary purification and extraction of the target protein. However, the high concentration of 8% PEG has too strong a precipitation ability, resulting in the loss of a large amount of Rubisco through co-precipitation, and a significant reduction in the amount of target protein remaining in the supernatant, which is not conducive to further purification and extraction.

[0050] Comparative Example 2 Compared to Example 1, the PEG concentration in the second precipitation was adjusted to 14%, and the results... Figure 10.

[0051] In the secondary precipitation, according to native PAGE, after secondary precipitation with the three PEG concentrations (8%, 10%, and 15%), the Rubisco holoenzyme (approximately 540 kDa) all showed a single main band in the 440–669 kDa range. Comparing the three: the band in the 8% PEG group was correctly positioned but relatively lightly stained; the band in the 10% PEG group was clear and concentrated, without obvious tailing or diffusion; the band in the 15% PEG group was the darkest stained but slightly diffused at the upper edge, suggesting that although the protein content was high, there might be slight aggregation or nonspecific co-precipitation.

[0052] SDS-PAGE results further revealed differences at the subunit level. Under all three conditions, characteristic Rubisco bands appeared at approximately 55 kDa (large subunit LSu) and approximately 15 kDa (small subunit SSu). The key difference was that the large subunit (55 kDa) and small subunit (10–15 kDa) bands were the clearest and sharpest in the 10% PEG group, with a harmonious ratio between the two subunits, indicating that Rubisco was not only abundant at this concentration but also relatively pure. Both large and small subunit bands were identifiable in the 8% PEG group, but the band intensity was significantly weaker than in the 10% group, suggesting that PEG precipitation at this concentration resulted in lower Rubisco separation. The staining depth of the large subunit bands in the 15% PEG group was similar to that in the 10% group, but the small subunit bands were relatively weaker, and there was a significant increase in bands of other proteins below 40 kDa, indicating that while excessively high PEG concentrations can increase the total protein precipitation, they sacrifice selectivity, leading to increased co-precipitation of non-target proteins.

[0053] In summary, a 10% PEG concentration is the optimal condition for secondary precipitation of Rubisco. At this concentration, the large and small subunit bands in the SDS-PAGE are the clearest, directly demonstrating that Rubisco is abundant and its subunit structure is intact, effectively removing impurities while ensuring high Rubisco recovery.

[0054] Comparative Example 3 Compared to Example 1, no TCEP, MgCl2, or glycerol were added during the pyrolysis process, resulting in... Figure 11 The electrophoresis results clearly demonstrate that the addition of lysis buffer (TCEP, MgCl2, and glycerol) significantly improves the protein extraction efficiency of the samples. The samples with added lysis buffer are significantly better than the samples without added lysis buffer (-) in terms of protein band integrity, quantity, and resolution.

[0055] The results of Native PAGE (non-denaturing polyacrylamide gel electrophoresis) showed that the samples (+) with lysis buffer containing TCEP, MgCl2, and glycerol exhibited clear and concentrated protein bands between 440 kD and 669 kD. This is highly consistent with the molecular weight of the native oligomer of Rubisco holoenzyme (L8S8, approximately 540-560 kD), indicating that the lysis buffer effectively maintained the native conformation and complex assembly state of the protein. In contrast, the samples (-) without lysis buffer containing TCEP, MgCl2, and glycerol showed significantly weakened and diffused bands in the same region, and more impurities appeared in the low molecular weight regions such as 66 kD and 45 kD. This indicates that the lack of lysis buffer did not adequately protect the protein, resulting in a reduced yield of the target protein.

[0056] The SDS-PAGE (denaturing polyacrylamide gel electrophoresis) results showed that the lysate with TCEP, MgCl2, and glycerol exhibited two clear main bands near approximately 55 kD and 15 kD, respectively. This is highly consistent with the molecular weights of the Rubisco large subunit (LSu, 53.4 kDa) and small subunit (SSu, 20.5 kDa) identified by mass spectrometry previously. Considering that the apparent molecular weight of proteins in SDS-PAGE may deviate slightly from the theoretical value, it is reasonable for 55 kD to correspond to LSU and 15 kD to correspond to SSu. In addition, there was a weaker band near approximately 70 kD, which has also been identified as Rubisco LSu based on the previous mass spectrometry results. This band may appear here because some of the large subunit and small subunit have bound together. In contrast, although the LSU and SSu bands could also be observed in the lysis buffer (-) without TCEP, MgCl2 and glycerol, the bands were generally shallow and the resolution was poor. In addition, there was obvious tailing and aggregation in the high molecular weight region above 130 kD, indicating that the protein extraction efficiency was low and there was a serious protein aggregation problem.

[0057] In summary, the addition of TCEP, MgCl2, and glycerol to the lysis buffer is crucial for the effective extraction of Rubisco from tobacco leaves. It not only significantly improves the extraction efficiency and yield of protein but also better maintains the natural oligomeric structure of the Rubisco holoenzyme (the intact complex of 440-669 kD in Native PAGE). At the same time, it can also obtain clearer and purer subunit bands (LSu approximately 55 kD and SSu approximately 15 kD) under denaturing conditions (SDS PAGE). In contrast, lysis buffer samples without TCEP, MgCl2, and glycerol result in insufficient cell lysis, leading to low protein release, increased degradation, and aggregation, which seriously affects the purity and quality of the protein.

[0058] Comparative Example 4 Compared to Example 1, the sucrose concentration gradient was replaced with a continuous sucrose gradient (5%-30%), otherwise it remained the same as Example 1. The results are as follows: Figure 12 As shown.

[0059] The results clearly demonstrate the significant shortcomings of continuous sucrose gradients (5%-30%) in Rubisco purification. Compared to the previous discontinuous gradients (20%-40%-60%), there was a significant decrease in separation efficiency, resolution, and final product purity. Native PAGE results showed extremely pale target bands, indicating a very small amount of the complete Rubisco holoenzyme complex obtained after purification, with most target proteins failing to be effectively enriched during centrifugation. SDS PAGE results also showed very pale bands for the large subunit (approximately 55 kD), further confirming the low yield of target proteins, while the small subunit (approximately 15 kD) band was completely absent. This suggests that in a continuous low-concentration sucrose environment, the Rubisco holoenzyme not only suffered significant loss but also underwent severe depolymerization, leading to the detachment or degradation of small subunits from the complex, preventing effective enrichment. Absorbance scans showed significantly lower main peak intensity and a wider, shorter peak shape with the same protein loading, indicating a lower protein concentration per unit volume and poor overall purification. In summary, continuous gradients of 5%-30% cannot effectively "capture" and enrich high molecular weight Rubisco holoenzymes due to insufficient upper density limit, lack of interfacial focusing effect and stepwise screening mechanism. This results in a large amount of target protein penetrating the gradient or being lost through depolymerization. The final purified product is not only small in quantity but also structurally incomplete (small subunits are missing). It is far inferior to discontinuous gradients of 20%-40%-60% in obtaining high concentration, high purity and structurally complete Rubisco holoenzyme complexes.

[0060] Comparative Example 5 Compared to Example 1, the sucrose concentration gradient was replaced with a discontinuous sucrose gradient of 10%-20%-40%, while the rest remained the same as in Example 1. The results are as follows: Figure 13 As shown.

[0061] This result further verifies the decisive influence of the upper limit of the density of the discontinuous sucrose gradient on the purification effect of Rubisco. Although the 10%-20%-40% gradient has the interfacial focusing advantage of the discontinuous gradient, it is obviously insufficient because the highest density layer is only 40%.

[0062] The Native PAGE results showed that the target band was pale, indicating a low recovery rate of the complete Rubisco holoenzyme complex. The SDS PAGE results also showed a pale band for the large subunit (approximately 55 kD), confirming a low yield of the target protein. The small subunit (approximately 15 kD) band was almost invisible, suggesting partial depolymerization of the Rubisco holoenzyme during centrifugation, with the small subunit detaching from the complex. The absorbance scans showed that, with the same protein loading, the intensity of the main peak in the 10%-20%-40% discontinuous gradient was low, indicating a low protein enrichment concentration and significant band broadening, resulting in insufficient separation resolution.

[0063] In summary, while the discontinuous gradient of 10%-20%-40% provides a certain focusing effect through the step-like interface, the highest density layer of 40% is still insufficient to effectively block the large molecular weight Rubisco holoenzyme (L8S8, approximately 540-560 kD). This results in some target proteins penetrating the bottom layer or undergoing structural dissociation under high centrifugal force, ultimately leading to a low amount of purified product, which is far inferior to the enrichment and extraction effect of 20%-40%-60% on Rubisco holoenzyme.

[0064] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for extracting Rubisco protein, characterized in that, Includes the following steps: Step 1: Provide the raw material extract containing the Rubisco protein to be extracted; Step 2: Add polyethylene glycol to the raw material extract to a final concentration of 0.01-0.05 g / mL, then centrifuge and collect the supernatant; Step 3: Add polyethylene glycol to the supernatant to a final concentration of 0.08-0.15 g / mL, and then centrifuge to obtain Rubisco protein precipitate.

2. The Rubisco protein extraction method according to claim 1, characterized in that, The polyethylene glycol is selected from at least one of PEG4000, PEG6000 or PEG8000; In step two, polyethylene glycol is added to a final concentration of 0.05 g / mL; in step three, polyethylene glycol is added to the supernatant to a final concentration of 0.10 g / mL.

3. The Rubisco protein extraction method according to claim 1, characterized in that, The centrifugation conditions are as follows: temperature: 0–10℃, centrifugal force: 10000–12000 g, time: 10 min–40 min, pH range: 6.5–8.

5.

4. The Rubisco protein extraction method according to claim 1, characterized in that, It also includes purification by density gradient centrifugation after resuspending Rubisco protein precipitate.

5. The Rubisco protein extraction method according to claim 4, characterized in that, The density gradient centrifugation is sucrose density gradient ultracentrifugation, with a sucrose gradient range of 0.2-0.6 g / mL; or a nonlinear sucrose concentration gradient of 0.2-0.4-0.6 g / mL is used during sucrose density gradient ultracentrifugation, with a centrifugal force of 100,000-200,000 g and a centrifugation time of 6-24 hours.

6. The Rubisco protein extraction method according to claim 1, characterized in that, The raw material extract contains: pH buffer, Mg 2+ Reducing agents, protease inhibitors, and glycerol; the pH of the raw material extract is 7.0–8.8; The pH buffer solution is selected from at least one of the following: Tris-HCl buffer system, PBS buffer system, phosphate buffer system, and HEPES buffer system. Mg 2+ The concentration is 5 mM to 20 mM; The reducing agent includes at least one of tris(2-carboxyethyl)phosphine, dithiothreitol, and β-mercaptoethanol; the concentration of the reducing agent is 1 mM to 5 mM. The protease inhibitors include at least one of benzyl sulfonyl fluoride, ethylenediaminetetraacetic acid, and leucopeptide; the concentration of the protease inhibitor is 1 mM to 5 mM.

7. The Rubisco protein extraction method according to claim 1, characterized in that, The raw material extract is obtained from plant tissue or from microbial or cell culture systems expressing Rubisco protein; and / or The plant tissue is a leaf from tobacco, spinach, Arabidopsis, rice, or wheat; and / or Polyvinylpyrrolidone is added during the extraction process.

8. The Rubisco protein extraction method according to claim 4, characterized in that, The method further includes purifying the Rubisco protein after density gradient centrifugation using at least one of gel filtration chromatography, ion exchange chromatography, or affinity chromatography.

9. A Rubisco protein sample obtained by any one of claims 1 to 8.

10. A method for analyzing Rubisco proteins, characterized in that, include: Rubisco protein samples were prepared using any one of the methods described in claims 1 to 8; The Rubisco protein sample was then analyzed using cryo-electron microscopy or X-ray crystallography.

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