A method for in vitro preparation of a rhodobacter cs cobt and cs cobn protein complex

Abstract

The application provides a preparation method of a Rhodobacter sphaeroides CsCobT and CsCobN protein complex in vitro, and steps are as follows: constructing an expression vector pET-28a(+)‑Cs CobN , pET-28a(+)‑SUMO‑Cs CobT ; expressing and purifying His-CsCobN protein; preparing a non-tagged CsCobT protein; mixing the purified His-CsCobN protein and the non-tagged CsCobT protein to form a protein complex; separating the complex through affinity chromatography; and refining the complex through gel filtration chromatography. The application adopts a strategy of double vectors, step-by-step purification, in-vitro assembly and multi-dimensional verification, can obtain a CsCobT and CsCobN protein complex with high purity and uniform conformation, and the experimental process can be accurately controlled, the result is specific, and provides high-quality protein materials for subsequent molecular mechanism research and application development of cobalt-chelating enzyme.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and protein engineering technology, and in particular to an in vitro preparation method of a protein complex of Rhodococcus spp. CsCobT and CsCobN. Background Technology

[0002] Vitamin B12 (cobalamin) is an essential nutrient for maintaining the normal physiological functions of humans, animals, and some microorganisms, and plays a key role in critical metabolic processes such as methyl transfer and nucleic acid synthesis. Cobalt chelate enzyme, as the core enzyme in the vitamin B12 biosynthesis pathway, is responsible for inserting cobalt ions into tetrapyrrole precursor compounds, which is a crucial step in the formation of the vitamin B12 active site.

[0003] Based on differences in their living environments, cobalt chelates are mainly divided into two categories: anaerobic and aerobic. Rhodopseudomonas aeruginosa (…) Cereibacter sphaeroides As a typical aerobic bacterium, its cobalt chelate is an aerobic multi-subunit metal chelate, consisting of a heterotrimer composed of the large subunit CsCobN, the medium subunit CsCobT, and the small subunit CsCobS. Sequence alignment analysis revealed varying degrees of sequence similarity between the CsCobN, CsCobT, and CsCobS subgenes and the three magnesium chelate subunits ChlH, ChlD, and ChlI, which are related to chlorophyll synthesis, suggesting a common evolutionary origin.

[0004] Currently, the gene sequences of each subunit of the cobalt chelate enzyme in Rhodopseudomonas spp. have been resolved, and bioinformatics analysis predicts that they can collectively constitute a biologically functional cobalt chelate enzyme. However, research on the direct interaction mechanism between the subunits and efficient in vitro assembly methods remains significantly insufficient, and existing techniques have several shortcomings: First, current research largely relies on in vivo co-expression strategies, constructing multiple subunit genes in the same vector and introducing them into host cells for expression. Under this method, the expression ratio of each subunit is difficult to precisely control due to the cellular environment, easily leading to the formation of inactive protein aggregates. Furthermore, the resulting complex is easily contaminated with other intracellular nucleic acid and protein components, making it impossible to accurately determine whether the interaction between subunits is a direct and specific binding, and hindering subsequent in-depth molecular mechanism research. Second, in vitro research methods lack rigorous control and validation systems; when crude extracts containing the target protein are directly mixed and incubated, impurities in the system can easily lead to contamination. Proteins can easily interfere with the interpretation of experimental results, leading to false positives or false negatives. For enzyme complexes like cobalt chelates, which may depend on metal ions and ATP, the presence of extraneous proteins can severely hinder the elucidation of their functional mechanisms. Third, there is a lack of assessment of the non-specific adsorption capacity of the subunits themselves. When using affinity chromatography to study protein interactions, if it is not verified beforehand whether each subunit has the ability to non-specifically bind to the affinity medium, it cannot be ruled out that the elution of the target protein in subsequent experiments is due to its own column attachment rather than the formation of a specific complex, which makes many research conclusions lack credibility. Fourth, the verification methods for complexes are limited. Most studies only stay at the level of affinity chromatography or immunoprecipitation, lacking effective confirmation of the conformational homogeneity and apparent molecular weight of the complex in solution, making it difficult to rule out the interference of non-specific aggregation or loose binding, and failing to prove that the obtained complex has the native conformation and biological functional activity.

[0005] Therefore, developing a method for the stable and efficient in vitro preparation of Rhodopseudomonas globulinus subunit protein complexes, and establishing a rigorous technical system including negative controls and multi-dimensional verification, is of great scientific significance and practical application value for elucidating the molecular mechanism of aerobic biosynthesis of vitamin B12 and promoting the research and application development of cobalt chelate enzymes. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide an in vitro preparation method for CsCobT and CsCobN protein complexes from Rhodococcus pluvialis. This method adopts a core strategy of expressing the complexes separately with dual vectors, stepwise purification, in vitro assembly, and multidimensional verification. This method can obtain high-purity, conformationally uniform CsCobT and CsCobN protein complexes. Moreover, the experimental process can be precisely controlled, and the results are highly specific. This provides high-quality protein materials for subsequent molecular mechanism research and application development of cobalt chelases.

[0007] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: An in vitro preparation method for a complex of CsCobT and CsCobN proteins from Rhodococcus globosum includes the following steps: (1) Construction of expression vector: Using molecular cloning technology, Cs CobN The gene was ligated into the pET-28a (+) vector, and Cs was added. CobT The gene was ligated into the pET-28a(+)-SUMO vector, and the recombinant plasmid pET-28a(+)-Cs was obtained. CobN pET-28a(+)-SUMO-Cs CobT ; (2) Expression and purification of His-CsCobN protein: pET-28a(+)-Cs CobN The protein was transformed into Escherichia coli, induced to express, and then subjected to sonication, Ni-NTA affinity chromatography, and gel filtration chromatography to obtain purified His-CsCobN protein. (3) Preparation of tagless CsCobT protein: pET-28a(+)-SUMO-Cs CobT The protein was transformed into E. coli and induced to express. The His-SUMO-CsCobT fusion protein was obtained by Ni-NTA affinity chromatography. SUMO enzyme was added to the fusion protein for digestion. The digestion product was subjected to Ni-NTA affinity chromatography again, and the flow-through was collected to obtain the tagless CsCobT protein. The tagless CsCobT protein was purified by gel filtration chromatography to obtain the purified tagless CsCobT protein. (4) In vitro assembly: The purified His-CsCobN protein was mixed with the untagged CsCobT protein and incubated to form a protein complex; (5) Affinity chromatography separation of the complex: The assembly product of step (4) was subjected to Ni-NTA affinity chromatography. After washing with buffer, the bound components were eluted with gradient imidazole elution buffer and the eluent was collected. (6) Gel filtration chromatography to purify the complex: After concentrating the elution fraction from step (5), gel filtration chromatography is performed to collect the elution peak fraction and obtain a stable and uniform CsCobT and CsCobN protein complex.

[0008] As one of the preferred embodiments of the present invention, in step (1), Cs CobN Genes, Cs CobT The nucleotide sequences of the gene are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the Cs CobN The gene was inserted between the Nde I and Xho I restriction sites in the pET-28a(+) vector, Cs CobT The gene was inserted between the BamHI and XbaI restriction sites of the pET-28a(+)-SUMO vector.

[0009] As one of the preferred embodiments of the present invention, in steps (2) and (3), the *Escherichia coli* is *Escherichia coli*. BL21 (DE3) .

[0010] As one of the preferred embodiments of the present invention, in steps (2) and (3), the inducing agent for expression is IPTG, with a final IPTG concentration of 0.4 mM, and the expression is induced at the bacterial culture OD. 600 When the concentration reaches 0.6~0.8, add an inducing agent and induce culture at 16℃ for 20h.

[0011] As one of the preferred embodiments of the present invention, in steps (2) to (5), the lysis / binding buffer for Ni-NTA affinity chromatography is 20mM Tris-HCl pH7.5 and 500mM NaCl, or 20mM Tris-HCl pH7.5 and 150mM NaCl; elution is performed using a gradient of 20mM, 200mM, and 500mM imidazole elution buffers, and the fraction eluted by 200mM imidazole is collected.

[0012] As one of the preferred embodiments of the present invention, in steps (2) to (6), the gel filtration chromatography uses a HiLoad™ 16 / 600 Superdex™ 200pg chromatography column, and the mobile phase buffer is 20mM Tris-HCl pH7.5 and 150mM NaCl.

[0013] As one of the preferred embodiments of the present invention, in step (3), the mass ratio of SUMO enzyme to His-SUMO-CsCobT fusion protein is 1:20, and the enzyme is digested overnight at 4°C.

[0014] As one of the preferred embodiments of the present invention, in step (4), the purified His-CsCobN protein and the unlabeled CsCobT protein are mixed at a molar ratio of 1:1 and incubated at 4°C for 2 hours to form a protein complex.

[0015] The advantages of this invention compared to the prior art are: (1) This invention adopts the overall strategy of "expression by two carriers, stepwise purification, in vitro assembly, and multidimensional verification". By preparing high-purity His-CsCobN and unlabeled CsCobT proteins in vitro, and using the characteristic of the unlabeled CsCobT that has no non-specific adsorption to nickel affinity chromatography medium as a key negative control, it can be strongly proved that the binding of CsCobN and CsCobT is a specific interaction, fundamentally eliminating false positive interference and significantly improving the accuracy and credibility of experimental conclusions.

[0016] (2) The present invention uses nickel ion affinity chromatography, SUMO protein cleavage to remove the tag and fine gel filtration chromatography to purify the CsCobT-CsCobN protein complex with high purity and strong uniformity. The gel filtration chromatography results show that the complex has a single symmetrical elution peak and the peak position is significantly forward compared with the monomer protein, which proves that the complex has a stable and uniform native conformation in solution, effectively solving the problems of easy aggregation, non-uniform conformation and single verification method of the complex in the prior art.

[0017] (3) This invention has successfully established a method for in vitro reconstruction of the core complex of cobalt chelate enzyme, providing key, mass-producible and homogeneous protein materials for in-depth research on the assembly mechanism, catalytic mechanism (such as the molecular details of cobalt ion insertion) of aerobic cobalt chelate enzymes (CobN / CobT / CobS), as well as their evolutionary relationship with magnesium chelate enzymes (ChlH / ChlD / ChlI), which are key enzymes in chlorophyll synthesis.

[0018] (4) The natural conformational protein complexes prepared by this method can be used for basic enzymatic studies such as enzyme kinetics and metal ion dependence, as well as for high-resolution structural analysis such as X-ray crystal diffraction and cryo-electron microscopy. At the same time, they can provide important biological elements and research models for synthetic biology modification based on the vitamin B12 synthesis pathway, optimization of industrial strains and screening of drug targets, which have both scientific value and practical application potential. Attached Figure Description

[0019] Figure 1 Cs in Example 1 CobN Agarose gel electrophoresis image of gene PCR amplification products (in the figure, M: Marker; 1, 2: Cs) CobN Gene PCR amplification products). Figure 2 Cs in Example 1 CobT Agarose gel electrophoresis image of gene PCR amplification products (in the figure, M: Marker; 1, 2, 3: Cs). CobT Gene PCR amplification products). Figure 3 It is the recombinant plasmid pET-28a(+)-Cs in Example 1 CobN Agarose gel electrophoresis image of double enzyme digestion identification (in the figure, M: Marker; 1, 2, 3, 4: enzyme digestion products); Figure 4 It is the recombinant plasmid pET-28a(+)-SUMO-Cs in Example 1 CobT Agarose gel electrophoresis image of double enzyme digestion identification (in the figure, M: Marker; 1, 2, 3, 4: enzyme digestion products); Figure 5This is an SDS-PAGE image of the His-CsCobN protein trial expression in Example 1 (in the image, M: Marker; pET-28a(+)-Cs CobN The expressed protein is 123 kDa in size. BL21(DE3) (can be expressed heterologously) Figure 6 This is an SDS-PAGE image of His-CsCobN protein purified by Ni-NTA affinity chromatography in Example 1 (in the image, M: Marker; 200mM imidazole protein is relatively pure and has a high expression level). Figure 7 shows the His-CsCobN protein gel filtration chromatography peak chromatogram and the corresponding SDS-PAGE chromatogram in Example 1 (Figure A is the chromatogram; Figure B is the SDS-PAGE chromatogram). Figure 8 This is an SDS-PAGE image of the His-SUMO-CsCobT fusion protein in Example 1 (in the image, pET-28a(+)-SUMO-C sCobT exist BL21(DE3) (can be expressed heterologously) Figure 9 This is an SDS-PAGE image of the His-SUMO-CsCobT fusion protein purified by Ni-NTA affinity chromatography in Example 1; Figure 10 This is an SDS-PAGE image of SUMO digestion and label-free CsCobT protein isolation in Example 1; Figure 11 These are the chromatograms and corresponding SDS-PAGE images of the label-free CsCobT protein gel filtration chromatography in Example 1. Figure 12 This is an SDS-PAGE image of the interaction between CsCobN and CsCobT verified by His Pull-down in Example 1 (in the image, M: Marker; 1: CsCobT (no tag) protein purified before binding; 2: His-CsCobT (no tag) protein purified before binding; 3: In vitro assembly system of CsCobT and CsCobN proteins (before passing through a nickel column); 4: Flow-through buffer not bound to the nickel column; 5: 200 mM imidazole elution buffer). Figure 13 This is a comparison of the peaks of the CsCobN-CsCobT complex and the monomer protein in gel filtration chromatography in Example 1. Detailed Implementation

[0020] 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. In the following embodiments, unless otherwise specified, all experimental methods are implemented in accordance with conventional technical means in the art; the reagents, carriers, strains, tool enzymes and other raw materials used are all commercially available conventional products.

[0021] Example 1 This embodiment describes an in vitro preparation method for a complex of CsCobT and CsCobN proteins from Rhodococcus spp., comprising the following steps: 1. Constructing an expression carrier Recombinant expression plasmid pET-28a(+)-Cs was constructed using molecular cloning technology. CobN pET-28a(+)-SUMO-Cs CobT The specific steps are as follows: (1) Regarding the existing publicly available Rhodopseudomonas spp. ( Cereibacter sphaeroides ) CobN Genes and CobT Gene analysis and codon optimization were performed to obtain optimized Cs. CobN Gene (shown in SEQ ID NO.1) and Cs CobT The gene (shown as SEQ ID NO.2) was used in subsequent experiments.

[0022] (2) According to Cs CobN Gene-specific primers (shown in SEQ ID NO.3 and SEQ ID NO.4) were designed using Cs-containing primers synthesized by the biotechnology company. CobN The gene plasmid was used as a template for PCR amplification to obtain... Cs CobN gene fragment. Using the same method, based on Cs CobT Gene sequence-specific primers (shown in SEQ ID NO.5 and SEQ ID NO.6) were designed to target Cs-containing compounds synthesized by the company. CobT The gene plasmid was used as a template for PCR amplification to obtain the CsCobT gene fragment.

[0023] The PCR amplification system consisted of 50 μL of the following components: 25 μL of 2×PhantaMax Master Mix, 1 μL of dNTP Mix, 1 μL of plasmid template containing the target gene, 2 μL each of upstream and downstream specific primers (10 μM), 1 μL of PhantaMax DNA Polymerase, and 18 μL of ddH2O. The PCR amplification program was as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, and 72℃ extension for 1 min 50 s (CsCo). bT) or 3 minutes 20 seconds (Cs) CobN ), a total of 30 cycles; final extension at 72℃ for 5 min; storage at 4℃. Cs CobN With Cs CobT Because gene lengths differ, the extension time during amplification varies. Specific primers are synthesized by commercial sequencing companies, and primer design must ensure that the amplified product contains recognition sequences at both ends corresponding to enzyme restriction sites.

[0024] Cs obtained by PCR amplification CobN Gene PCR products, Cs CobT The gene PCR products were verified by agarose gel electrophoresis. Figure 1 , Figure 2 After confirming that the amplified band is the same size as the target gene, the target band is excised and the target gene fragment is recovered and purified using an agarose gel DNA recovery kit.

[0025] (3) The purified Cs were recovered CobN The gene was double-digested with Nde I and Xho I restriction endonucleases to extract Cs. CobT The gene was double-digested with BamHI and XbaI restriction endonucleases. Simultaneously, the commercially available pET-28a(+) vector and pET-28a(+)-SUMO vector (Wuhan Miaoling Biotechnology Co., Ltd., "Miaoling Plasmid Platform", product code: P0028) were double-digested with the corresponding restriction endonucleases. The digestion reaction volume was 20 μL, including: 17 μL of target gene / vector, 0.5 μL of each restriction endonuclease, and 2 μL of 10x FDG buffer. Digestion conditions: 37℃ for 1 h. After digestion, the fragments were recovered and purified to obtain the linearized vector and the double-digested target gene fragment.

[0026] (4) Ligate the double-digested target gene to the corresponding digested vector. The ligation system includes: 6 μL target gene, 2.5 μL vector, 1 μL T4 DNA buffer, and 0.5 μL T4 DNA Ligase. Ligation conditions: 25℃ for 3 h. Transform the ligation product into Escherichia coli. TOP10 Competent cells were plated on LB agar plates containing kanamycin and incubated overnight at 37°C.

[0027] (5) Select single clones and inoculate them into LB liquid medium containing kanamycin for culture, and extract plasmid DNA. Take the recombinant plasmid and perform double digestion with the corresponding restriction endonuclease for identification. The digestion products are analyzed by agarose gel electrophoresis. The electrophoresis results show ( Figure 3 , Figure 4 If the size of the enzyme digestion fragment is consistent with the expected target gene fragment and vector fragment, it is preliminarily determined to be a positive clone.

[0028] (6) The recombinant plasmids that tested positive for double enzyme digestion were sent to a sequencing company for sequencing. Sequencing results showed that Cs... CobN The gene was correctly inserted between the Nde I and Xho I restriction sites in the pET-28a (+) vector. CobT The gene was correctly inserted between the BamHI and XbaI restriction sites in the pET-28a (+)-SUMO vector, with the correct reading frame and no mutations. Therefore, the recombinant plasmid pET-28a (+)-Cs was successfully obtained. CobN pET-28a (+)-SUMO-Cs CobT .

[0029] 2. Expression and purification of His-CsCobN protein (1) Before large-scale expression, it is necessary to test the expression level of the protein. After confirming that the protein can be stably expressed in BL21(DE3), large-scale culture can be carried out. First, the correctly identified recombinant plasmid pET-28a(+)-Cs CobN Transform to BL21(DE3) Single clones were picked and cultured in 4 mL LB liquid medium at 37°C with shaking until OD reached. 600 Once the concentration reaches 0.6-0.8, 200 μL of bacterial culture is taken as a pre-induction control. The remaining bacterial culture is then treated with IPTG (final concentration 0.4 mM), and expression is induced for 4 hours. 200 μL of the induction culture is then taken as the post-induction sample. Protein expression is detected by SDS-PAGE electrophoresis. Figure 5 ).

[0030] (2) The identified recombinant clone strain expressing the protein was inoculated into 10 mL of LB liquid medium and cultured overnight at 37°C. The next day, the inoculation was increased to 1 L of TB liquid medium (to obtain richer nutrients and higher cell density to increase the total yield of the target protein) at a 1:100 ratio and cultured at 37°C with shaking until OD. 600 When the concentration reaches 0.6~0.8, add IPTG to a final concentration of 0.4mM, induce culture at 16℃ for 20h, and then collect the bacterial cells by centrifugation.

[0031] (3) Perform Ni-NTA affinity chromatography, the specific steps of which are as follows: 1) Resuspend the collected bacterial cells in lysis buffer (20mM Tris-HCl pH 7.5, 500mM NaCl). This operation should be performed on ice.

[0032] 2) Use an ultrasonic cell disruptor to disrupt cells. The operating procedure is: 300W, disruption for 4 seconds, interval for 9 seconds, two ultrasonic cycles, one cycle for 30 minutes. During disruption, ensure that the centrifuge tubes are in an ice-water mixture and the cell disruption solution is at a low temperature. The disruption program can be adjusted according to the actual situation.

[0033] 3) The crushed sample was centrifuged at 4°C and 12,000 rpm for 40 min in a floor-standing refrigerated centrifuge to separate the supernatant and precipitate, and samples were taken for testing.

[0034] 4) Rinse the Ni-NTA gel for 2 column volumes with deionized water, and then rinse the Ni-NTA gel for 2 column volumes with buffer (with the same composition as the lysis buffer) to equilibrate the affinity column.

[0035] 5) Add the balanced Ni-NTA affinity gel to the supernatant after centrifugation and incubate slowly at 4°C for 40 minutes to allow the protein to fully bind with the Ni-NTA gel.

[0036] 6) After incubation, the protein solution is slowly added to the affinity chromatography column. After two flow-throughs, the sample is taken and discarded.

[0037] 7) Elute with three gradients of imidazole elution buffer (20mM, 200mM, and 500mM) and collect the elution buffers in separate tubes. Use Quick Start™ Bradford protein staining solution (BIO-RAD) for each gradient until the staining solution changes color, then move on to the next gradient.

[0038] SDS-PAGE electrophoresis was performed on samples obtained in each step, and the results are as follows: Figure 6 As shown in the figure, the results indicate that a high-salt environment with 500mM NaCl can shield the non-specific electrostatic adsorption between contaminating proteins and the nickel column medium, thereby reducing the non-specific binding of the nickel column. Proteins are purer and have higher expression levels under 200mM imidazole. The 200mM imidazole elution buffer was subsequently concentrated for further purification.

[0039] (4) Perform gel filtration chromatography, the specific steps of which are as follows: Collect the 200 mM imidazole elution fraction from the previous steps, concentrate it to 2 ml, and pass it through a molecular sieve (gel filtration chromatography). The molecular sieve chromatography column was a HiLoad™ 16 / 600 Superdex™ 200 pg column, and the molecular sieve buffer was 20 mM Tris-HCl pH 7.5 and 150 mM NaCl. Collect the corresponding samples according to the peak positions and perform SDS-PAGE electrophoresis for detection.

[0040] The molecular sieve peak diagram and electrophoresis detection diagram of His-CsCobN protein are as follows: Figure 7As shown, the peak position is approximately 63 mL, and according to the column calibration formula, it is approximately 127 kDa, indicating that the His-CsCobN protein in the solution exists in a monomeric state. SDS-PAGE showed that the protein sample was relatively pure, and 61 mL of the protein below the peak was collected for the preparation of the complex of the two subsequent proteins.

[0041] 3. Preparation and verification of label-free CsCobT protein and its non-specific adsorption (1) pET-28a(+)-SUMO-CsCobT was transformed into Escherichia coli BL21 (DE3) After stable expression was confirmed, large-scale induction of expression was performed, and bacterial cells were collected. The cells were then sonicated and centrifuged to obtain the supernatant. The supernatant was loaded onto a nickel ion affinity chromatography column, and after binding, eluted with imidazole-containing buffer. The elution peak fraction was collected using 200 mM imidazole elution buffer to obtain the His-SUMO-CsCobT fusion protein. The specific steps and methods are the same as those described in the previous section on "Expression and Purification of His-CsCobN Protein," and the corresponding results are as follows: Figure 8 , 9 As shown.

[0042] Subsequent steps, including enzyme digestion and incubation, all use buffers of 20mM Tris-HCl pH 7.5 and 150mM NaCl, with salt concentrations close to physiological salts. This helps maintain the protein's native conformation, ensures accurate localization in FPLC, and promotes specific interactions.

[0043] (2) SUMO enzyme was added to the purified His-SUMO-CsCobT fusion protein at a protein:enzyme ratio of 20:1 for overnight digestion to remove the His-SUMO tag. The digestion product was then loaded back onto a nickel ion affinity chromatography column, and the flow-through fraction was collected. At this point, the removed His-SUMO tag and SUMO enzyme protein were adsorbed onto the column, while the target protein... Cs Because CobT does not carry any affinity tag, it flows directly into the permeate solution, and the flow-through component is collected, thus obtaining a tagless product. Cs CobT protein. SUMO enzyme and other components attached to the column were eluted with 500 mM imidazole. SDS-PAGE results are shown below. Figure 10 As shown.

[0044] This step also constitutes a crucial negative control experiment: SDS-PAGE analysis showed that the CsCobT protein recovered during flow-through was not adsorbed onto the nickel column, demonstrating that the CsCobT protein itself has no non-specific binding ability to the nickel affinity chromatography medium. This result provides important evidence for the reliability of subsequent pull-down experiments.

[0045] (3) After concentrating the collected unlabeled CsCobT protein, gel filtration chromatography was performed. The specific steps and methods were the same as those described in the "Expression and Purification of His-CsCobN Protein". The fraction corresponding to 61 mL of the main peak was collected to obtain unlabeled CsCobT protein with high purity and uniform conformation. The chromatogram and corresponding SDS-PAGE results are shown in the figure. Figure 11 As shown.

[0046] 4. In vitro assembly The purified His-CsCobN protein and the untagged CsCobT protein were mixed at a 1:1 molar ratio and incubated at 4°C for 2 hours to allow them to fully interact and form a complex.

[0047] 5. His Pull-down verification of the interaction between CsCobN and CsCobT The complex obtained from the above steps was loaded onto a nickel ion affinity chromatography column for His pull-down assay. The column was thoroughly washed with buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl) to remove unbound and non-specifically adsorbed proteins, followed by elution with imidazole-containing buffer. The elution peaks were collected and analyzed by SDS-PAGE.

[0048] Verification Principle: In this step, His-CsCobN serves as the bait protein, and the untagged CsCobT serves as the prey protein. Since step 3 above has rigorously demonstrated that the untagged CsCobT protein itself has no non-specific adsorption capacity for the nickel column, its appearance in the eluent must depend on forming a stable complex with His-CsCobN and being specifically captured by the latter. If SDS-PAGE shows the simultaneous presence of both CsCobN and CsCobT protein bands in the eluent, it proves that there is a direct in vitro interaction between the two.

[0049] SDS-PAGE results are as follows Figure 12 As shown, the presence of two protein bands, CsCobN and CsCobT, in the 200mM elution buffer indicates a direct in vitro interaction between them.

[0050] 6. Molecular sieve chromatography confirmation of the complex The eluted fraction of the complex obtained in step 5 was concentrated and then subjected to molecular sieve chromatography. The eluted peak fraction was collected and compared with the peak positions of the His-CsCobN protein and the untagged CsCobT protein alone.

[0051] Verification criteria: If the peak position of the complex sample is significantly forward compared to the two monomeric proteins, it proves that CsCobN and CsCobT form a stable and homogeneous complex, and that the complex has a native folded conformation in solution.

[0052] Using the same gel adsorption column (HiLoad™ 16 / 600 Superdex™ 200 pg) as when purifying His-CsCobN and CsCobT (no tag) monomeric proteins, the peak position of the CsCobN / CsCobT complex was found to be 57 mL, which is significantly forward compared to the two monomers (His-CsCobN 63 mL, CsCobT 61 mL). Figure 13 This further confirms that CsCobN and CsCobT can form a stable and homogeneous complex.

[0053] In summary, this embodiment successfully established an in vitro preparation method for the CsCobT and CsCobN protein complex from Rhodococcus pluvialis using a strategy of "separate expression on dual carriers, stepwise purification, in vitro assembly, and multidimensional verification." This method first efficiently expresses and purifies high-purity His-CsCobN protein and tagless CsCobT protein separately. Then, through precise in vitro assembly, affinity chromatography separation, and gel filtration chromatography purification, a conformationally homogeneous and highly stable CsCobT and CsCobN protein complex was finally obtained. Simultaneously, the non-specific adsorption verification of the tagless CsCobT protein, His pull-down assay, and multidimensional verification by gel filtration chromatography fully demonstrated the specificity and homogeneity of the complex, providing high-quality protein materials for subsequent research on the molecular mechanism of cobalt chelases and their application development.

[0054] The above description is merely 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 in vitro preparation of a complex of CsCobT and CsCobN proteins from Rhodococcus pluvialis, characterized in that, Includes the following steps: (1) Construction of expression vector: Using molecular cloning technology, Cs CobN The gene was ligated into the pET-28a (+) vector, and Cs was added. CobT The gene was ligated into the pET-28a(+)-SUMO vector, and the recombinant plasmid pET-28a(+)-Cs was obtained. CobN pET-28a(+)-SUMO-Cs CobT ; (2) Expression and purification of His-CsCobN protein: pET-28a(+)-Cs CobN The protein was transformed into Escherichia coli, induced to express, and then subjected to sonication, Ni-NTA affinity chromatography, and gel filtration chromatography to obtain purified His-CsCobN protein. (3) Preparation of tagless CsCobT protein: pET-28a(+)-SUMO-Cs CobT The protein was transformed into E. coli and induced to express. The His-SUMO-CsCobT fusion protein was obtained by Ni-NTA affinity chromatography. SUMO enzyme was added to the fusion protein for digestion. The digestion product was subjected to Ni-NTA affinity chromatography again, and the flow-through was collected to obtain the tagless CsCobT protein. The tagless CsCobT protein was purified by gel filtration chromatography to obtain the purified tagless CsCobT protein. (4) In vitro assembly: The purified His-CsCobN protein was mixed with the untagged CsCobT protein and incubated to form a protein complex; (5) Affinity chromatography separation of the complex: The assembly product of step (4) was subjected to Ni-NTA affinity chromatography. After washing with buffer, the bound components were eluted with gradient imidazole elution buffer and the eluent was collected. (6) Gel filtration chromatography to purify the complex: After concentrating the elution fraction from step (5), gel filtration chromatography is performed to collect the elution peak fraction and obtain a stable and uniform CsCobT and CsCobN protein complex.

2. The preparation method according to claim 1, characterized in that, In step (1), Cs CobN Genes, Cs CobT The nucleotide sequences of the gene are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the Cs CobN The gene was inserted between the Nde I and Xho I restriction sites in the pET-28a(+) vector, Cs CobT The gene was inserted between the BamHI and XbaI restriction sites of the pET-28a(+)-SUMO vector.

3. The preparation method according to claim 1, characterized in that, In steps (2) and (3), the *E. coli* is *Escherichia coli*. BL21 (DE3) .

4. The preparation method according to claim 1, characterized in that, In steps (2) and (3), the inducing agent for expression is IPTG, with a final IPTG concentration of 0.4 mM, and the concentration is determined by the bacterial culture OD. 600 When the concentration reaches 0.6~0.8, add an inducing agent and induce culture at 16℃ for 20h.

5. The preparation method according to claim 1, characterized in that, In steps (2) to (5), the lysis / binding buffer for Ni-NTA affinity chromatography is 20mM Tris-HCl pH7.5 and 500mM NaCl, or 20mM Tris-HCl pH7.5 and 150mM NaCl; elution is performed using a gradient of 20mM, 200mM, and 500mM imidazole elution buffers, and the fraction eluted by 200mM imidazole is collected.

6. The preparation method according to claim 1, characterized in that, In steps (2) to (6), the gel filtration chromatography uses a HiLoad™ 16 / 600 Superdex™ 200pg chromatography column, and the mobile phase buffer is 20mM Tris-HCl pH7.5 and 150mM NaCl.

7. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of SUMO enzyme to His-SUMO-CsCobT fusion protein is 1:20, and the enzyme is digested overnight at 4°C.

8. The preparation method according to claim 1, characterized in that, In step (4), the purified His-CsCobN protein and the unlabeled CsCobT protein are mixed at a 1:1 molar ratio and incubated at 4°C for 2 hours to form a protein complex.

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