A solubility-promoting tag cd3 and its use in protein soluble expression

Abstract

The present application relates to a kind of solubility-promoting tags CD3 and its application in protein soluble expression.CD3 is the 3rd color structure domain (Chromodomain 3, CD3) of cpSRP43, and the present application aims to explore the application of CD3 domain to improve protein solubility and expression amount.The present application proves that the efficiency of solubility-promoting tags CD3 in dissolving recombinant protein is equivalent to that of traditional fusion tags maltose-binding protein (MBP), glutathione-S-transferase (GST), small ubiquitin-like modifier protein (SUMO) and thioredoxin (TrxA).It can not only make recombinant protein soluble efficiently like traditional fusion tags, but also is small enough, which does not affect the activity of recombinant protein and does not bring additional burden to the host.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biosynthesis technology, specifically a low molecular weight solubilizing tag CD3 and its application in the soluble expression of proteins. Background Technology

[0002] Recombinant proteins are widely produced in a variety of organisms. Among these, *E. coli* is frequently used as an expression platform for high-level production of exogenous proteins due to its rapid growth, well-developed genetic characterization, and abundant available molecular engineering tools. When attempting to express human proteins in *E. coli*, approximately 25% are insoluble. Even among soluble proteins, approximately 50–75% fail to exhibit the expected activity.

[0003] Several strategies have been developed to soluble recombinant proteins. One widely accepted strategy is to fuse these proteins with soluble tags, including maltose-binding protein (MBP), glutathione S-transferase (GST), small ubiquitin-like modified protein (SUMO), and thioredoxin (TrxA). This fusion typically not only improves the solubility of the target protein but also facilitates purification. However, these tags should be removed before use because they may sterically hinder the active site of the recombinant protein. Furthermore, these tags may impose an additional burden on the host as they consume additional cellular resources. Therefore, there is a pressing need for a novel fusion tag that can soluble recombinant proteins as efficiently as traditional fusion tags, while being small enough not to affect the activity of the recombinant protein or impose an additional burden on the host. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for expressing a target protein using the lysing tag CD3.

[0005] An application of a solubilizing tag in the soluble expression of proteins, wherein the amino acid sequence of the solubilizing tag is shown in SEQ ID NO.1 in the sequence listing, and the target protein for soluble protein expression is a red fluorescent protein mKeima-Red tagged with Avitag and tobacco plaque virus protease TEV-S219V.

[0006] Furthermore, the nucleotide sequence of the gene encoding the solubilizing tag is shown in SEQ ID NO.2 of the sequence listing.

[0007] A method for expressing a target protein using the lysosomal tag CD3 includes the following steps: S1. The fusion tag encoding gene sequence is linked to the target protein encoding gene to obtain a fusion protein expression unit, and the fusion protein expression unit is inserted into an expression plasmid to construct a fusion protein expression vector; wherein, the fusion tag is CD3, and its encoding gene sequence is shown in SEQ ID NO. 2; the target protein is Avitag-tagged red fluorescent protein Avitag-mKeima-Red and tobacco plaque virus protease TEV-S219V; S2. Transform the fusion protein expression vector obtained in step S1 into a host strain to obtain a transformed host strain, and culture the transformed host strain to induce expression of the fusion protein, wherein the fusion protein is a target protein containing a CD3 tag.

[0008] A fusion protein comprising the aforementioned solubilizing tag and the target protein.

[0009] Furthermore, the gene encoding the solubilizing tag is located upstream of the gene encoding the target protein.

[0010] A fusion protein expression vector containing the gene encoding the aforementioned fusion protein.

[0011] A recombinant bacterial strain containing the aforementioned fusion protein expression vector.

[0012] Furthermore, the strain is Escherichia coli.

[0013] The beneficial effects of this invention are: (1) The efficiency of the solubilizing tag CD3 in dissolving recombinant proteins is comparable to that of traditional fusion tags such as maltose-binding protein (MBP), glutathione-S-transferase (GST), small ubiquitin-like modified protein (SUMO), and thioredoxin (TrxA). It can solubilize recombinant proteins as efficiently as traditional fusion tags, and it is small enough that it does not affect the activity of recombinant proteins or impose an additional burden on the host.

[0014] (2) The solubilizing tag CD3 is as efficient as the traditional fusion tag in solubilizing recombinant proteins and does not need to be removed when using fusion recombinant proteins. It is expected to promote the high-level production of functional recombinant proteins suitable for multiple uses and drive the progress of the protein production industry in bacterial systems. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0016] Figure 1This is an agarose gel electrophoresis image of the recombinant plasmids constructed by plasmid PCR and double enzyme digestion verification of each tag fusion with the Avitag-mKeima-Red gene in Example 1 of this invention. Figure 2 This is an agarose gel electrophoresis image of the recombinant plasmid constructed by plasmid PCR and double enzyme digestion verification of each tag fusion gene with TEV-S219V in Example 2 of the present invention. Figure 3 This is an SDS-PAGE analysis diagram of the expression of each tag with the Avitag-mKeima-Red fusion protein in Example 3 of the present invention; Figure 4 This is an SDS-PAGE analysis diagram of the expression of each tag with the TEV-S219V fusion protein in Example 3 of the present invention; Figure 5 The figures show the effects of each tag in Example 3 of the present invention on the overall protein expression level and solubility of Avitag-mKeima-Red; where A is the effect of each tag in Example 3 of the present invention on the relative expression level of Avitag-mKeima-Red protein; and B is the effect of each tag in Example 3 of the present invention on the solubility of Avitag-mKeima-Red protein. Figure 6 The following diagrams illustrate the effects of each tag in Example 3 of this invention on the overall protein expression level, solubility, and relative expression level of TEV-S219V; where A represents the effect of each tag on the protein expression level of TEV-S219V in Example 3 of this invention; B represents the effect of each tag on the solubility of TEV-S219V in Example 3 of this invention; and C represents the effect of each tag on the relative expression level of TEV-S219V protein in Example 3 of this invention. Figure 7 This is a graph showing the electrophoretic analysis results of protein expression in Example 4 of the present invention, where each fusion protein cleaves the TEV protease substrate. Detailed Implementation

[0017] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0018] In the description of this invention, it should be understood that "a plurality of" means two or more, unless otherwise explicitly specified.

[0019] The present invention will now be further illustrated with specific examples. The following embodiments are only for explaining the present invention and do not constitute a limitation thereof. The test samples and test procedures used in the following embodiments include the following: If the specific experimental conditions are not specified in the embodiments, they are generally performed according to conventional conditions or the conditions recommended by the reagent company; unless otherwise specified, the reagents, consumables, etc. used in the following embodiments can be obtained commercially.

[0020] In this invention, cpSRP43 is a key protein in plant and algal chloroplasts and a highly specific, non-ATP-dependent molecular chaperone that can effectively reverse the aggregation of its substrate membrane proteins. CD3 is the third chromomodomain (CD3) of cpSRP43, and this invention aims to explore the application of the CD3 domain in improving protein solubility and expression levels.

[0021] Therefore, this invention aims to protect the application of CD3 as a solubilizing tag in the soluble expression of proteins.

[0022] In this invention, the molecular weight of the solubilizing tag CD3 is 5.6 KD, and its amino acid sequence is shown in SEQ ID NO.1 of the sequence listing; the nucleotide sequence of the gene encoding the solubilizing tag CD3 is shown in SEQ ID NO.2 of the sequence listing.

[0023] It should be noted that the *E. coli* BL21(DE3) and the original plasmid pET28a(+) used in the experiments of this invention were both purchased from Wuhan Qingke Biotechnology Co., Ltd., and grown on conventional LB medium. The plasmid extraction, vector cloning, and protein expression and purification procedures are described in *Molecular Cloning: A Laboratory Manual* (4th Edition).

[0024] <Example 1> The construction of the recombinant expression vector, including its gene information, primer information, and PCR reaction conditions, are shown in Tables 1-4.

[0025] The gene sequence information used in this embodiment is shown in Table 1.

[0026] Table 1

[0027] The primer information used in this embodiment is shown in Table 2.

[0028] Table 2

[0029] The amounts of each component added to the PCR reaction system in the examples are shown in Table 3.

[0030] Table 3

[0031] The PCR reaction program settings for this embodiment are shown in Table 4.

[0032] Table 4

[0033] In this embodiment, protein fusion was performed using CD3, GST, MBP, SUMO, and TrxA tags, and various recombinant expression vectors were constructed. The main steps are as follows: S1. Construct a recombinant expression vector of Avitag-mKeima-Red without any fusion tag as a negative control. The steps are as follows: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Hind After the III restriction site is ligated to the Avitag tag (Avi tag), add to both ends. SaI I and Not I. Restriction site, specifically introduced at the 5′ end of the Avitag tag. SaI I. Enzyme digestion sequence and introduction of the 3′ end of mKeima-Red protein Not I. The enzyme sequence was digested and then ligated into the pET28a(+) vector to obtain the recombinant expression vector pET28a-Avitag-mKeima-Red (SEQ ID NO.13); S2. Construction of recombinant expression vectors containing different fusion tags: After linking an Avitag tag to the N-terminus of the wild-type mKeima-Red protein, CD3, GST, MBP, SUMO, and TrxA were used as fusion tags. The sequences of the fusion tags, Avitag, and mKeima-Red were simultaneously ligated and inserted into the pET28a(+) vector using PCR and enzyme digestion ligation methods, resulting in the recombinant expression vectors pET28a-CD3-Avitag-mKeima-Red, pET28a-GST-Avitag-mKeima-Red, pET28a-MBP-Avitag-mKeima-Red, pET28a-SUMO-Avitag-mKeima-Red, and pET28a-TrxA-Avitag-mKeima-Red.

[0034] The restriction enzyme site is introduced at the 5′ end of the solubilization tag. Nde I restriction site, introduced at the 5′ end of the Avitag tag. SaI I. Enzyme cleavage site, introduction of the 3′ end of the mKeima-Red protein Not Ⅰ. Enzyme cleavage site.

[0035] Positive verification of recombinant expression vectors: The recombinant expression vectors constructed above were transformed into E. coli BL21(DE3) competent cells, plasmid DNA was extracted, and PCR was performed followed by agarose gel electrophoresis for verification. The pET28a-Avitag-mKeima-Red recombinant expression vector without any tag was used as a negative control. The recombinant expression vectors that were correctly verified by PCR were then sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing verification (e.g.,...). Figure 1 As shown), by Figure 1 It can be confirmed that the correct recombinant expression vectors were obtained.

[0036] Figure 1 The information for each lane is as follows: M represents the protein marker (1 kb); lanes 1-2 are respectively: pET28a-Avitag-mKeima-Red plasmid, Sal I + Not I double digestion (742 bp, Avitag-mKeima-Red); lanes 3-5 are respectively: pET28a-MBP-Avitag-mKeima-Red plasmid, Sal I + Not I double digestion (742 bp, Avitag-mKeima-Red), Nde I + Not I double digestion (1845 bp, MBP-Avitag-mKeima-Red); lanes 6-9 are respectively: pET28a-CD3-Avitag-mKeima-Red plasmid, Sal I + Nde I double digestion (153 bp, CD3), Sal I + Not I double enzyme digestion (742bp, Avitag-mKeima-Red), Nde I + Not I double digestion (895 bp, CD3-Avitag-mKeima-Red); lanes 10-12 are respectively: pET28a-GST-Avitag-mKeima-Red plasmid, Sal I + Not I double digestion (742 bp, Avitag-mKeima-Red), Nde I + Not I double digestion (1396 bp, GST-Avitag-mKeima-Red); lanes 13-15 are respectively: pET28a-TrxA-Avitag-mKeima-Red plasmid, Sal I + NotI double digestion (742 bp, Avitag-mKeima-Red), Nde I + Not I double digestion (1069 bp, TrxA-Avitag-mKeima-Red); lanes 16-18 are respectively: pET28a-SUMO-Avitag-mKeima-Red plasmid, Sal I + Not I double digestion (742 bp, Avitag-mKeima-Red), Nde I + Not I double digestion (1048 bp, SUMO-Avitag-mKeima-Red).

[0037] <Example 2> The construction of the recombinant expression vector, including its gene information, primer information, and PCR reaction conditions, are shown in Tables 2-6.

[0038] Table 5

[0039] Table 6

[0040] In this embodiment, CD3, GST, MBP, SUMO, and TrxA tags were fused with tobacco plaque virus protease (TEV-S219V), and a 6×His tag was added to the C-terminus of TEV-S219V for affinity purification. Various recombinant expression vectors were constructed, and the main steps are as follows: S1. Construct a recombinant expression vector for TEV-S219V without any fusion tag as a negative control. The steps are as follows: Add fusion tags to both ends of the TEV-S219V protein. SaI I and Not I. Enzyme cleavage site, specifically introduced at the 5′ end of the TEV-S219V protein. SaI I. Enzyme cleavage site, introduction of the 3′ end of the TEV-S219V protein Not I. The restriction enzyme sites were then ligated into the pET28a(+) vector to obtain the recombinant expression vector pET28a-TEV-S219V; S2. Construction of recombinant expression vectors containing different fusion tags: After ligating a 6×His tag to the C-terminus of the TEV-S219V protein, CD3, GST, MBP, SUMO, and TrxA were used as fusion tags. The fusion tags and the sequence of TEV-S219V were ligated simultaneously using PCR and enzyme digestion ligation methods and inserted into the pET28a(+) vector to obtain the recombinant expression vectors pET28a-CD3-TEV-S219V, pET28a-GST-TEV-S219V, pET28a-MBP-TEV-S219V, pET28a-SUMO-TEV-S219V, and pET28a-TrxA-TEV-S219V, respectively.

[0041] It should be noted that the five tags CD3, GST, MBP, SUMO, and TrxA were fused to the N-terminus of the TEV-S219V protein and then inserted into the pET28a plasmid.

[0042] Specifically, the introduction of the restriction enzyme site involves introducing it at the 5′ end of the solubilization tag. Nde I restriction site, introduced at the 5′ end of the TEV-S219V protein. SaI I. Restriction site: Introduction of the 3′ end of the 6×His tag Not I. Enzyme cleavage sites; Gly-Ala linkers were used between the lysis tag coding sequence and the TEV-S219V protein sequence, as well as between the TEV-S219V protein sequence and the 6×His tag sequence. These are all existing technologies, and the specific experimental operation methods will not be described in detail in this invention.

[0043] Positive verification of recombinant expression vectors: The recombinant expression vectors constructed above were transformed into E. coli BL21(DE3) competent cells, plasmid DNA was extracted, and PCR was performed followed by agarose gel electrophoresis for verification. The pET28a-TEV-S219V recombinant expression vector without any tag was used as a negative control. The recombinant expression vectors that were correctly verified by PCR were then sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing verification (e.g.,...). Figure 2 As shown), by Figure 2 It can be confirmed that the correct recombinant expression vectors were obtained.

[0044] Figure 2 The information for each lane is as follows: M is the protein marker (1 kb); lanes 1-4 are respectively: pET28a-GST-TEV-S219V plasmid PCR (879 bp). Sal I + Nde I double digestion (654 bp, GST), Sal I + NotI double enzyme digestion (708bp, TEV-S219V) Nde I + Not I double digestion (1362 bp, GST-TEV-S219V); lanes 5-8 were respectively: pET28a-MBP-TEV-S219V plasmid PCR (879 bp). Sal I + Nde I double digestion (1103 bp, MBP), Sal I + Not I double digestion (708 bp, TEV-S219V), Nde I + Not I double digestion (1811 bp, MBP-TEV-S219V); lanes 9-13 are respectively: pET28a-TrxA-TEV-S219V plasmid, plasmid PCR, Sal I + Nde I double digestion (364 bp, TrxA), Sal I + Not I double digestion (893 bp, TEV-S219V), Nde I + Not I double digestion (1257 bp, TrxA-TEV-S219V); lanes 14-18 respectively: pET28a-SUMO-TEV-S219V plasmid, plasmid PCR, Sal I + Nde I double digestion (306 bp, SUMO) Sal I + Not I double digestion (893 bp, TEV-S219V), Nde I + Not I double digestion (1201 bp, SUMO-TEV-S219V); lanes 19-21 respectively: pET28a-TEV-S219V plasmid, plasmid PCR, Sal I + Nde I double digestion (893 bp, TEV-S219V); lanes 22-24 are respectively pET28a-CD3-TEV-S219V plasmid, plasmid PCR, and... Sal I + Not I. Double enzyme digestion (708 bp, TEV-S219V).

[0045] <Example 3> Expression of fusion proteins.

[0046] The recombinant expression vectors constructed in Examples 1-2 were transformed into competent Escherichia coli BL21(DE3) cells. After single colonies grew, single colonies containing the fusion protein were selected and cultured in 5 mL LB liquid medium with 5 μL of 0.1 g / mL kanamycin added. The cultures were incubated at 37°C and 180 rpm until OD500. 600 When the bacterial culture reaches approximately 0.6, 500 μL of the culture is transferred to 50 mL of LB liquid medium, and 50 μL of 0.1 g / mL kanamycin is added. The culture is then incubated at 37°C and 180 rpm until the OD value is reached. 600 When the concentration was around 0.6, IPTG was added to the culture medium to a final concentration of 0.4 mM, and the fusion protein expression was induced for 12 h at 28°C and 150 rpm.

[0047] The expressed bacterial culture was lysed using an ultrasonic homogenizer. 200 μL of the lysed whole bacterial culture was transferred to a 1.5 mL sterile centrifuge tube and stored. The remaining bacterial culture was stored at 4°C and centrifuged at 10,000 g for 15 min. 200 μL of the supernatant was transferred to a 1.5 mL sterile centrifuge tube and stored. The remaining supernatant was filtered through a 0.2 μm filter membrane for subsequent purification. The bacterial pellet was resuspended in 6 mL of pre-chilled PBS buffer, and 200 μL of the pellet was transferred to a 1.5 mL EP tube and stored. The stored samples were then used for SDS-PAGE identification.

[0048] The molecular weights of the 10 fusion proteins were determined and are shown in Table 7.

[0049] Table 7

[0050] After SDS-PAGE analysis, the results of various tags promoting the expression of fusion proteins are as follows: Figures 3-4 As shown. Figure 3 In the swimming pool, lanes 1-2 represent the supernatant and precipitate of *E. coli* transformed with pET28a-Avitag-mKeima-Red, respectively; lanes 3-4 represent the supernatant and precipitate of *E. coli* transformed with pET28a-CD3-Avitag-mKeima-Red, respectively; lanes 5-6 represent the supernatant and precipitate of *E. coli* transformed with pET28a-GST-Avitag-mKeima-Red, respectively; lanes 7-8 represent the supernatant and precipitate of *E. coli* transformed with pET28a-TrxA-Avitag-mKeima-Red, respectively; lanes 9-10 represent the supernatant and precipitate of *E. coli* transformed with pET28a-MBP-Avitag-mKeima-Red, respectively; and lanes 11-12 represent the supernatant and precipitate of *E. coli* transformed with pET28a-SUMO-Avitag-mKeima-Red, respectively. Figure 3 It was found that the Avitag-mKeima-Red protein (molecular weight 30 kDa) without any added tags was not expressed at 28°C for 12 h; the band sizes of the fusion proteins CD3-Avitag-mKeima-Red, GST-Avitag-mKeima-Red, MBP-Avitag-mKeima-Red, TrxA-Avitag-mKeima-Red and SUMO-Avitag-mKeima-Red in the electrophoresis gel were consistent with expectations, indicating that the lysing tags of the present invention can increase the expression level of Avitag-mKeima-Red protein.

[0051] Figure 4 In the swimming pool, lanes 1-2 represent the supernatant and precipitate of *E. coli* transformed with pET28a-TEV-S219Vd, respectively; lanes 3-4 represent the supernatant and precipitate of *E. coli* transformed with pET28a-CD3-TEV-S219V, respectively; lanes 5-6 represent the supernatant and precipitate of *E. coli* transformed with pET28a-GST-TEV-S219V, respectively; lanes 7-8 represent the supernatant and precipitate of *E. coli* transformed with pET28a-TrxA-TEV-S219V, respectively; lanes 9-10 represent the supernatant and precipitate of *E. coli* transformed with pET28a-MBP-TEV-S219V, respectively; and lanes 11-12 represent the supernatant and precipitate of *E. coli* transformed with pET28a-SUMO-TEV-S219V, respectively. Figure 4 It can be seen that the expression level of TEV-S219V protein (molecular weight 28 kDa) without any added tag was low in the supernatant under the conditions of 28°C and 12 h. The band sizes of the fusion proteins CD3-TEV-S219V, GST-TEV-S219V, MBP-TEV-S219V, TrxA-TEV-S219V and SUMO-TEV-S219V in the electrophoresis gel were consistent with expectations, and the tags could increase the expression level of TEV-S219V protein.

[0052] The expression levels of the fusion protein in this invention were analyzed using Image J. The results are as follows: Figures 5-6 As shown in the figure ( (This indicates a p-value < 0.0001). (From...) Figures 5-6 It can be seen that the total expression of fusion proteins with CD3 tags has a significant advantage, and their solubility is also significantly higher than that of the control group without solubilizing tags and other fusion proteins with solubilizing tags.

[0053] <Example 4> The core objective of this example is to compare the effects of five different tags—CD3, GST, MBP, SUMO, and TrxA—fused to the TEV-S219V protein on the cleavage activity of the TEV-S219V protease itself, thereby evaluating the effect of the fusion tag CD3 on the activity of the target protein.

[0054] The TEV-S219V protein needs to be purified to obtain a tagged TEV-S219V protease sample (TEV-Protease) for subsequent assays of tag detachability and hydrolytic activity. The gene information involved in this embodiment is shown in Table 8. The reagent kit used for protein purification is Ni NTAAgarose Beads 6FF (QS01002) from Jiangsu Qianzhusong Biotechnology Co., Ltd. The TEV-S219V protein is tagged with a His tag. The protein purification operation is a prior art technique, and its specific experimental procedures will not be described in detail in this embodiment.

[0055] Table 8

[0056] The specific steps are as follows: S1. Construction of TEV protease substrate: After sequentially ligating the sequences of mNeonGreen, TEVsit (SEQ ID NO.26), and Nluc, add to both ends... Nde I and Sal Specifically, an Nde I restriction site was introduced at the 5′ end of the mNeonGreen sequence and a Sal I restriction site was introduced at the 3′ end of the Nluc sequence to construct the TEV protease substrate (mNeonGreen-TEVsit-Nluc, SEQ ID NO.27).

[0057] S2. Using mNeonGreen-TEVsit-Nluc as a substrate, the TEV protease substrate mNeonGreen-TEVsit-Nluc was digested with the fusion proteins CD3-TEV-S219V, GST-TEV-S219V, MBP-TEV-S219V, TrxA-TEV-S219V, and SUMO-TEV-S219V from Example 4, respectively. TEV-S219V protease without a lysis-promoting tag was used as a control group. The protein expression results of each fusion protein and the control group were analyzed by 12% SDS-PAGE electrophoresis. Figure 7 ).

[0058] In this fusion protein, mNeonGreen is a fluorescent protein, TEVsit is the recognition site of the TEV-S219V protease, and Nluc is luciferase. When the fusion protein has TEV-S219V cleavage activity, it recognizes and cleaves the TEV site, thereby releasing the fluorescent protein mNeonGreen and the luciferase Nluc, resulting in fluorescence. The higher the cleavage activity, the more fluorescent protein mNeonGreen and luciferase Nluc are released, and the higher the concentration of the corresponding band on SDS-PAGE.

[0059] Specifically, in this embodiment, the cleavage system was determined to have a ratio of fusion protein to TEV protease substrate of 0.8:1. Under these conditions, the 70 μL cleavage system is shown in Table 9.

[0060] Table 9

[0061] Figure 7 The information for each lane is as follows: M: 10-130 kDa Marker; Lane 1: Substrate mNeonGreen-TEVsit-Nluc, where the molecular weight of mNeonGreen is 27 kDa and the molecular weight of Nluc is 23 kDa. Lane 2: Cleavage product after incubation of TEV-S219V Protease without solubilization tag for 4 h; Lanes 4, 6, 8, 10, and 12: Cleavage products after incubation of TEV Protease with solubilization tags CD3, GST, TrxA, MBP, and SUMO, respectively, for 4 h; Lane 3: Cleavage product after incubation of TEV Protease without solubilization tag for 6 h; Lanes 5, 7, 9, 11, and 13: Cleavage products after incubation of TEV Protease with solubilization tags CD3, GST, TrxA, MBP, and SUMO, respectively, for 6 h. The bands at 23 kDa in lanes 12 and 13 are contaminant protein bands from the purification of SUMO-TEV-S219V.

[0062] Depend on Figure 7It was found that the molecular weights of the cleavage products mNeonGreen and Nluc were approximately 27 kDa and 23 kDa, respectively. The TEV-S219V protease without the added solubilizing tag could also recognize and cleave the substrate, but its hydrolysis efficiency was significantly lower than that of the TEV-S219V protease with the added solubilizing tag, as indicated by the yield of hydrolyzed substrate (band depth). The fusion proteins CD3-TEV-S219V, MBP-TEV-S219V, and SUMO-TEV-S219V significantly increased the hydrolysis efficiency of the TEV protease substrate, and this efficiency increased with prolonged incubation time. This indicates that the addition of the CD3, MBP, and SUMO tags significantly improved the efficiency of TEV-S219V protein in hydrolyzing the substrate.

[0063] The above-disclosed embodiments are merely one or more preferred embodiments of this application and should not be construed as limiting the scope of this application. Those skilled in the art can understand that all or part of the processes for implementing the above embodiments and equivalent changes made in accordance with the claims of this application still fall within the scope of this application.

Claims

1. An application of a solubilizing tag in the soluble expression of proteins, characterized in that, The amino acid sequence of the solubilizing tag is shown in SEQ ID NO.1 in the sequence listing, and the protein is either the red fluorescent protein mKeima-Red or the tobacco plaque virus protease TEV-S219V.

2. The application as described in claim 1, characterized in that, The nucleotide sequence of the gene encoding the solubilizing tag is shown in SEQ ID NO.2 in the sequence listing.

3. A method for expressing a target protein using the solubilization tag CD3, characterized in that, Includes the following steps: S1. The fusion tag encoding gene sequence is linked to the target protein encoding gene to obtain a fusion protein expression unit, and the fusion protein expression unit is inserted into the expression plasmid to construct a fusion protein expression vector; wherein, the fusion tag is CD3, and its encoding gene sequence is shown in SEQ ID NO. 2; The target protein is either the Avitag-tagged red fluorescent protein mKeima-Red or the tobacco plaque virus protease TEV-S219V; Furthermore, when the target protein is tobacco plaque virus protease TEV-S219V, the lysis tag encoding gene sequence and the TEV-S219V protein sequence are linked using a Gly-Ala linker. When the target protein is a red fluorescent protein mKeima-Red tagged with Avitag, the fusion protein after adding the lysing tag encoding gene sequence is CD3-Avitag-mKeima-Red; S2. Transform the fusion protein expression vector obtained in step S1 into a host strain to obtain a transformed host strain, and culture the transformed host strain to induce expression of the fusion protein, wherein the fusion protein is a target protein containing a CD3 tag.

4. A fusion protein, characterized in that, It includes the solubilizing tag and the target protein as described in claim 3.

5. The fusion protein according to claim 4, characterized in that, The gene encoding the solubilization tag is located upstream of the gene encoding the target protein.

6. A fusion protein expression vector, characterized in that, The gene encoding the fusion protein as described in claim 4 or 5.

7. A recombinant bacterial strain, characterized in that, The expression vector containing the fusion protein as described in claim 6.

8. The recombinant strain according to claim 7, characterized in that, The strain in question is Escherichia coli.

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