SuperTEV protein expression construct for improving expression quantity of escherichia coli recombinant protein and expression method of SuperTEV protein expression construct
By adding VNP6 tags to the N-terminal of the recombinant protein in E. coli and combining it with low temperature culture, the abnormal protein folding and insufficient yield caused by intracellular molecular crowding were solved, and efficient and stable recombinant protein expression and activity were achieved.
Patent Information
- Application Number
- CN202510439092.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-09
- Publication Date
- 2025-06-24
AI Technical Summary
When E. coli is expressed in high-density proteins, it is easy to cause abnormal protein folding, aggregation and inclusion bodies due to intracellular molecules, which in turn reduces the activity and yield of recombinant proteins.
By adding VNP6 tags to the N-terminal of the recombinant protein and combining novel constructs and expression methods with low temperature culture, we induce the formation of vesicles or chesticoid structures that are conducive to protein folding and stable, reducing molecular crowding in cells and improving the correct folding rate and activity of recombinant proteins.
It significantly improves the expression amount and activity of recombinant proteins in E. coli, reduces the risk of protein aggregation and degradation, and achieves efficient and stable recombinant protein production.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a SuperTEV protein expression construct for improving the expression level of recombinant proteins in Escherichia coli and its expression method. Background Art
[0002] In recent years, with the increasing importance of recombinant proteins in the fields of biomedicine, industrial production, and basic research, various expression systems such as mammalian cells, yeasts, and Escherichia coli have been widely developed and utilized. Among them, Escherichia coli has become the preferred platform for industrial-scale recombinant protein production due to its short culture cycle, low cost, fast expression rate, and simple operation. However, since Escherichia coli is a prokaryote, there are significant differences in its intracellular biosynthesis environment compared to eukaryotic cells. Especially when the cell density is high and the expression level is fast, problems such as abnormal protein folding, non-specific aggregation, and inclusion body formation are likely to occur. These phenomena are closely related to intracellular molecular crowding, that is, under the competition of high concentrations of proteins and other biomacromolecules, the space between molecules is limited, affecting the correct folding and efficient functional realization of proteins, and thus reducing the activity and yield of the final product.
[0003] In Escherichia coli, high-density protein expression is often accompanied by the formation of organelle-like structures or vesicle-like structures, but these structures are not necessarily conducive to the correct folding and function of recombinant proteins. To overcome this bottleneck, there is an urgent need to develop more innovative strategies to alleviate molecular crowding by regulating the intracellular microenvironment.
[0004] Traditionally, in order to improve the expression efficiency and secretion level of specific recombinant proteins, it has been reported that the VNP6 peptide sequence is used to promote the generation of extracellular vesicles. The VNP6 tag can regulate cell signal transduction and vesicle secretion processes in some eukaryotic cells, and improve the extracellular distribution and stability of proteins by inducing the exocytic pathway. However, this strategy mainly focuses on using VNP6 to trigger the activation of the protein secretion mechanism by cells, and its research and application targets are concentrated on cell systems capable of complex secretion regulation, while paying insufficient attention to the important problem faced by prokaryotic microorganisms such as Escherichia coli - namely, molecular crowding caused by limited intracellular space.
[0005] In summary, how to effectively alleviate the problem of intracellular molecular crowding and improve the expression level of target proteins has become one of the urgent problems to be solved in this field. Summary of the Invention
[0006] To solve the above technical problems, the present invention provides a SuperTEV protein expression construct for increasing the expression level of recombinant proteins in Escherichia coli and its expression method. By adding a VNP6 tag to the N-terminus of the recombinant protein and combining it with a novel construct and expression method of low-temperature culture, the problem of insufficient protein expression caused by intracellular molecular crowding in the Escherichia coli expression system is successfully solved.
[0007] To achieve this purpose, the present invention adopts the following technical solutions:
[0008] In a first aspect, the present invention provides a SuperTEV protein expression construct for increasing the expression yield of recombinant proteins in Escherichia coli cells, and the SuperTEV protein expression construct includes a VNP6 tag coding sequence and a SuperTEV protein coding sequence.
[0009] As a commonly used biotechnological tool, the SuperTEV protein can rely on its specific recognition of the cleavage position between Q and S in the ENLYQSI sequence in the substrate, providing an accurate and controllable cleavage means for site-directed protein expression and subsequent processing. The present invention uses the VNP6 tag to induce the formation of vesicles or vesicle-like structures in Escherichia coli that are beneficial to protein folding and stability, and uses this peptide sequence to promote the concentration of intracellular vesicles, thereby reducing the overall intracellular molecular crowding effect while increasing the correct folding rate and activity of the recombinant protein. Combining the SuperTEV protein with the VNP6 tag strategy not only provides additional guarantee for the activity regulation of the protein in the later stage of expression, but also enables the system to achieve higher expression and processing efficiency in the face of the complex biochemical environment in Escherichia coli cells.
[0010] Preferably, the nucleic acid sequence of the VNP6 tag coding sequence includes the sequence shown in SEQ ID NO.1.
[0011] SEQ ID NO.1: ATGGATGTATTTAAAAAGGGATTCTCAATAGCAGATGA AGGTGTGGTGGGCGCAGTTGAGAAAACCGATCAGGGTGTGACCGAGGCC GCGGAAAAGACCAAAGAGGGTGTT.
[0012] Preferably, the nucleic acid sequence of the SuperTEV protein coding sequence includes the sequence shown in SEQ ID NO.2.
[0013] SEQ ID NO.2: GGCTCTGGTGAAAGCTTATTCAAGGGTCCGAGAGACT ACAATCCGATTAGCTCCAGCATTTGCCATTTGACGAACGAGAGCGATGGTCACACCACGTCGCTGTATGGCATCGGCTTTGGTCCGTTTATCATCACCAACAAGCACTTGTTCCGCCGTAATAACGGTACACTTGTGGTTCAGAGCCTCCATGGTGTTTTTAAGGTGAAGGACACCGCTACCCTGCAACAGCATTTGGTCGATGGCCGTGACATGATGATTATCCGCATGCCTAAGGACTTCCCACCATTCCCGCAAAAACTGAAATTTCGCGAGCCGCAACGTGAGGAACGTATTTGTCTGGTAACTACGAACTTCCAGGCGAAAAGCATGTCCAGCATGGTGTCCGACACCAGTTGCACGTTTCCGAGCGGTGATGGCACGTTTTGGAAACATTGGATTCAAACCAAAGACGGCCAGTGCGGCAATCCGCTGGTCAGCACCCGTGACGGCTTCATCGTTGGTATCCATTCTGCGAGCAATTTCACCAACACCAACAACTACTTCGCCTCCGTGCCGAAAAACTTCATGGAACTGCTGACCAACCAGGAGGCGCAGCAATGGGTTTCGGGCTGGCGTCTGAATGCGGATAGCGTTCTGTGGGGCGGCCACAAAGTGTTTATGGTTAAGCCGGAAGAGCCGGGTTCC。
[0014] The SuperTEV protein provided by the present invention is a self-optimized version of TEV protease, and its expression level and activity have been adjusted through previous experiments.
[0015] Preferably, the SuperTEV protein expression construct further comprises a His10 tag coding sequence and a StrepII tag coding sequence.
[0016] Preferably, the nucleic acid sequence of the StrepII tag coding sequence comprises the sequence shown in SEQ ID NO.3.
[0017] SEQ ID NO.3: TGGAGCCATCCGCAATTTGAAAAG.
[0018] Preferably, the SuperTEV protein expression construct sequentially comprises, from the 5'-end to the 3'-end: a VNP6 tag coding sequence, a His10 tag coding sequence, a SuperTEV protein coding sequence, and a StrepII tag coding sequence.
[0019] In the present invention, the His10 tag is used for subsequent protein purification and immunoassay, and the StrepII tag aids in protein purification and functional assay.
[0020] Preferably, the nucleic acid sequence of the SuperTEV protein expression construct comprises the sequence shown in SEQ ID NO.4.
[0021] SEQ ID NO.4: ATGGATGTATTTAAAAAGGGATTCTCAATAGCAGATGA AGGTGTGGTGGGCGCAGTTGAGAAAACCGATCAGGGTGTGACCGAGGCCGCGGAAAAGACCAAAGAGGGTGTTATGAGCGGCGGTGGTTCTGGCCATCACCATCACCACCACCACCACCACCACGGCTCTGGTGAAAGCTTATTCAAGGGTCCGAGAGACTACAATCCGATTAGCTCCAGCATTTGCCATTTGACGAACGAGAGCGATGGTCACACCACGTCGCTGTATGGCATCGGCTTTGGTCCGTTTATCATCACCAACAAGCACTTGTTCCGCCGTAATAACGGTACACTTGTGGTTCAGAGCCTCCATGGTGTTTTTAAGGTGAAGGACACCGCTACCCTGCAACAGCATTTGGTCGATGGCCGTGACATGATGATTATCCGCATGCCTAAGGACTTCCCACCATTCCCGCAAAAACTGAAATTTCGCGAGCCGCAACGTGAGGAACGTATTTGTCTGGTAACTACGAACTTCCAGGCGAAAAGCATGTCCAGCATGGTGTCCGACACCAGTTGCACGTTTCCGAGCGGTGATGGCACGTTTTGGAAACATTGGATTCAAACCAAAGACGGCCAGTGCGGCAATCCGCTGGTCAGCACCCGTGACGGCTTCATCGTTGGTATCCATTCTGCGAGCAATTTCACCAACACCAACAACTACTTCGCCTCCGTGCCGAAAAACTTCATGGAACTGCTGACCAACCAGGAGGCGCAGCAATGGGTTTCGGGCTGGCGTCTGAATGCGGATAGCGTTCTGTGGGGCGGCCACAAAGTGTTTATGGTTAAGCCGGAAGAGCCGGGTTCCTGGAGCCATCCGCAATTTGAAAAGTAA。
[0022] In a second aspect, the present invention provides a recombinant vector, which contains the SuperTEV protein expression construct described in the first aspect.
[0023] In a third aspect, the present invention provides a recombinant Escherichia coli cell, which contains the SuperTEV protein expression construct described in the first aspect and / or the recombinant vector described in the second aspect.
[0024] In a fourth aspect, the present invention provides a recombinant protein, the coding sequence of which includes the SuperTEV protein expression construct described in the first aspect.
[0025] Preferably, the amino acid sequence of the recombinant protein includes the sequence shown in SEQ ID NO.5.
[0026] SEQ ID NO.5: MDVFKKGFSIADEGVVGAVEKTDQGVTEAAEKTKEGV MSGGGSGHHHHHHHHHHGSGESLFKGPRDYNPISSSICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLVVQSLHGVFKVKDTATLQQHLVDGRDMMIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQAKSMSSMVSDTSCTFPSGDGTFWKHWIQTKDGQCGNPLVSTRDGFIVGIHSASNFTNTNNYFASVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEEPGSWSHPQFEK.
[0027] In a fifth aspect, the present invention provides a method for inducing the expression of SuperTEV protein in Escherichia coli cells, the method for inducing expression including: transferring the recombinant vector described in the second aspect into Escherichia coli cells, and inducing the expression of SuperTEV protein with IPTG at 10 - 20°C;
[0028] or the method for inducing expression including: inducing the expression of SuperTEV protein with IPTG at 10 - 20°C for the recombinant Escherichia coli cell described in the third aspect.
[0029] Different from the traditional use of VNP6 to promote the secretion of extracellular vesicles, the core concept of the present invention is to regulate the internal structure of cells through low temperature, optimize the microenvironment inside the Escherichia coli expression system, so that protein molecules under high expression load can obtain a more reasonable spatial distribution, and reduce the problems of misfolding and aggregation caused by molecular crowding.
[0030] The specific values of the above 10 - 20°C can be 10°C, 12°C, 14°C, 16°C, 18°C or 20°C, etc.
[0031] Preferably, the temperature for inducing expression is 14 - 18°C.
[0032] The specific values of 14-18°C described above can be 14°C, 15°C, 16°C, 17°C, 18°C, etc.
[0033] Preferably, the working concentration of the IPTG is 0.3-0.5 mM.
[0034] The specific values of 0.3-0.5 mM described above can be 0.3 mM, 0.35 mM, 0.38 mM, 0.4 mM, 0.42 mM, 0.45 mM, 0.5 mM, etc.
[0035] Preferably, the method for inducing expression specifically includes: transferring the recombinant vector described in the second aspect into Escherichia coli cells, picking a single colony into an LB liquid medium, culturing at 35-40°C, and when the OD 600 of the bacterial liquid is 0.6-0.8, adding IPTG to a final concentration of 0.3-0.5 mM, and inducing expression at 10-20°C for 10-20 h.
[0036] The specific values of 35-40°C described above can be 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.
[0037] The specific values of 0.6-0.8 described above can be 0.6, 0.65, 0.7, 0.75, 0.8, etc.
[0038] The specific values of 0.3-0.5 mM described above can be 0.3 mM, 0.35 mM, 0.38 mM, 0.4 mM, 0.42 mM, 0.45 mM, 0.5 mM, etc.
[0039] The specific values of 10-20°C described above can be 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, etc.
[0040] The specific values of 10-20 h described above can be 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, etc.
[0041] Other specific point values within the ranges of the above-mentioned values can be selected, and will not be elaborated one by one here.
[0042] Compared with the prior art, the present invention has the following beneficial effects:
[0043] The present invention provides a SuperTEV protein expression system for regulating intracellular vesicle formation based on the VNP6 tag. By inducing internal structural changes, it effectively alleviates the problem of molecular crowding inside cells, providing a brand-new technical path for improving the expression level, correct folding, and biological activity of target proteins. When expressed in Escherichia coli, a low-temperature (such as 16 °C) culture condition is adopted. Under this condition, the secretion of extracellular vesicles is not promoted, but the concentration of intracellular vesicles is facilitated, thereby preventing molecular crowding inside cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0044] Figure 1 It is a graph showing the protein expression results of Example 2 of the present invention.
[0045] Figure 2 It is a graph showing the protein purification results of Example 3 of the present invention.
[0046] Figure 3 It is a graph showing the enzyme activity test results of Example 4 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0047] To further elaborate on the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with examples and drawings. It can be understood that the specific embodiments described herein are only used to explain the present invention, rather than limiting the present invention.
[0048] For those not specifying specific techniques or conditions in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.
[0049] Example 1
[0050] In this example, a VNP6-His10-SuperTEV-StrepII protein expression construct was constructed, which sequentially included, from the 5'-end to the 3'-end: the VNP6 tag coding sequence (SEQ ID NO.1), the His10 tag coding sequence, the SuperTEV protein coding sequence (SEQ ID NO.2), and the StrepII tag coding sequence (SEQ ID NO.3). The nucleic acid sequence of the VNP6-His10-SuperTEV-StrepII protein expression construct included the sequence shown in SEQ ID NO.4, and the amino acid sequence of the obtained recombinant protein was as shown in SEQ ID NO.5.
[0051] Example 2
[0052] In this example, the protein expression construct prepared in Example 1 was introduced into the modified Escherichia coli. The LB medium was selected, and 0.4 mM IPTG was used to induce expression at 16 °C for 16 h. SDS-PAGE and Western Blot experiments were performed on the recombinant protein, and the results are as Figure 1 shown. The expression level of the recombinant protein VNP6-His10-SuperTEV-StrepII in the cells was significantly increased. Under the same culture conditions, the expression level of the recombinant protein in the control group of the His10-SuperTEV-StrepII construct without the VNP6 tag was significantly lower than that of the group with the VNP6 tag, further verifying the role of the VNP6 tag in intracellular vesicle concentration and protein expression enhancement in the method of the present invention.
[0053] Example 3
[0054] In this example, the recombinant protein VNP6-His10-SuperTEV-StrepII was purified by Ni-NTA affinity using the His10 tag. During the elution process, a gradient-increasing imidazole concentration could be used to ensure the high-purity acquisition of the target protein. The results of the SDS-PAGE experiment are as Figure 2 shown. 30 mM imidazole was used to wash away the proteins non-specifically bound to the Ni-NTA resin, and the target protein with high purity was successfully eluted through a gradient-increasing imidazole concentration from 250 mM (Elution1), 350 mM (Elution 2), 450 mM (Elution 3) to 500 mM (Elution 4).
[0055] Example 4
[0056] In this example, in order to verify whether the enzyme activity of the VNP6-His10-SuperTEV-Strep II protein is retained during the intracellular vesicle concentration process, a reporter system containing the SuperTEV specific substrate, namely VNP6-ENLYQSI-IL1Ra, was constructed. First, an enzymatic cleavage experiment was carried out using the purified VNP6-His10-SuperTEV-StrepII protein. In the reaction system, the SuperTEV protein specifically recognized the ENLYQSI sequence in the reporter substrate and carried out cleavage between Q and S. Subsequently, the reaction products were detected by SDS-PAGE electrophoresis, and cleavage fragments consistent with the expectations were observed. The overall test results showed that the expressed VNP6-His10-SuperTEV-StrepII protein had the expected enzymatic catalytic cleavage function.
[0057] The present invention significantly alleviates the problem of intracellular molecular crowding by introducing the VNP6 tag at the N-terminus of the recombinant protein and supplementing it with a low-temperature culture strategy, thereby protecting the recombinant protein from aggregation or degradation and significantly increasing its expression level. Specifically, the vesicle condensation effect induced by the VNP6 tag not only optimizes the intracellular microenvironment but also makes the protein folding and modification processes more efficient under low-temperature culture conditions, further ensuring the high activity and stability of the recombinant protein.
[0058] In addition, experimental data show that under low-temperature culture conditions (such as culturing at 16°C), the protein expression yield of this construct is significantly increased (by more than 100 times), and it has no negative impact on the activity of SuperTEV enzyme. Moreover, it shows better process adaptability in some reaction systems. The research results confirm that the VNP6 tag not only demonstrates unique advantages in protein localization and concentration but also plays an important role in the stable expression of thermosensitive and functional proteins. This method has practical application value in production, provides an efficient and reliable technical route for large-scale industrial production of recombinant proteins, and is expected to be further extended to the preparation of other proteins that are difficult to express efficiently.
[0059] In summary, the novel construct and expression method proposed in the present invention, which add the VNP6 tag at the N-terminus of the recombinant protein and combine it with low-temperature culture, successfully solve the problem of insufficient protein expression often caused by intracellular molecular crowding in the Escherichia coli expression system. By regulating protein distribution using the intracellular vesicle concentration effect, the risk of protein aggregation and degradation is greatly reduced, and efficient and stable production of recombinant proteins is achieved.
[0060] This method not only increases the expression level of the recombinant protein but also retains the enzyme cleavage activity of SuperTEV, showing great potential in industrial production. By introducing the intracellular vesicle regulation strategy into protein engineering and expression systems, the present invention expands the means of recombinant protein expression and provides a novel and effective technical solution to solve the bottleneck problems in traditional Escherichia coli expression systems. Further popularization and application of this technology will help improve the preparation efficiency of related biological products and the controllability of production processes, and promote the development of the biomedical and industrial biotechnology fields.
[0061] The applicant declares that the above description is only a specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and public scope of the present invention.
Claims
1. A SuperTEV protein expression construct for increasing the expression yield of recombinant proteins in Escherichia coli cells, characterized in that: The SuperTEV protein expression construct includes a VNP6 tag coding sequence and a SuperTEV protein coding sequence.
2. The SuperTEV protein expression construct according to claim 1, characterized in that: The nucleic acid sequence of the VNP6 tag encoding sequence includes the sequence shown in SEQ ID NO.1; Preferably, the nucleic acid sequence of the SuperTEV protein coding sequence includes the sequence shown in SEQ ID NO.
2.
3. The SuperTEV protein expression construct according to claim 1 or 2, characterized in that: The SuperTEV protein expression construct also includes a His10 tag coding sequence and a StrepII tag coding sequence; Preferably, the nucleic acid sequence of the StrepII tag encoding sequence includes the sequence shown in SEQ ID NO.
3.
4. The SuperTEV protein expression construct according to any one of claims 1 to 3, characterized in that: The SuperTEV protein expression construct includes, from the 5' end to the 3' end: VNP6 tag coding sequence, His10 tag coding sequence, SuperTEV protein coding sequence and StrepII tag coding sequence; Preferably, the nucleic acid sequence of the SuperTEV protein expression construct includes the sequence shown in SEQ ID NO.
4.
5. A recombinant vector, characterized in that: The recombinant vector contains the SuperTEV protein expression construct according to any one of claims 1 to 4.
6. A recombinant Escherichia coli cell, characterized in that The recombinant Escherichia coli cell contains the SuperTEV protein expression construct according to any one of claims 1 to 4 and / or the recombinant vector according to claim 5.
7. A recombinant protein, characterized in that The coding sequence of the recombinant protein includes the SuperTEV protein expression construct according to any one of claims 1 to 4.
8. The recombinant protein according to claim 7, characterized in that The amino acid sequence of the recombinant protein includes the sequence shown in SEQ ID NO.
5.
9. A method for inducing expression of SuperTEV protein in Escherichia coli cells, characterized in that: The inducible expression method comprises: transferring the recombinant vector of claim 5 into Escherichia coli cells, and inducing the expression of SuperTEV protein by IPTG at 10-20°C; Or the inducing expression method comprises: inducing the recombinant Escherichia coli cells according to claim 6 to express the SuperTEV protein using IPTG at 10-20°C.
10. The inducible expression method according to claim 9, characterized in that: The temperature for inducing expression is 14-18°C; Preferably, the working concentration of IPTG is 0.3-0.5 mM; Preferably, the inducing expression method specifically comprises: transferring the recombinant vector of claim 5 into Escherichia coli cells, picking a single clone colony into LB liquid culture medium, culturing at 35-40°C, and waiting for the bacterial solution OD 600 When the pH value is 0.6-0.8, add IPTG to a final concentration of 0.3-0.5 mM and induce expression at 10-20°C for 10-20 h.