Method for increasing protein expression quantity by enhancing formation of mRNA (messenger ribonucleic acid) ribosome complex

Abstract

The invention belongs to the technical field of biology, and relates to a method for increasing protein expression quantity by enhancing formation of mRNA (messenger ribonucleic acid) ribosome compounds. The method comprises the following steps: constructing a recombinant expression vector containing a first expression element of a target protein gene controlled by a first inducible promoter and a second expression element of a ribosome bS1 protein gene controlled by a second inducible promoter, introducing the recombinant expression vector into a recombinant bacterium, firstly inducing the expression of the target protein, and then inducing the expression of the ribosome bS1 protein, thereby obtaining the ribosome bS1 protein. Finally, the purpose of improving the expression of the target protein is achieved. Therefore, the invention can be used for improving the expression of the target protein and has wide application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for increasing the protein expression level by enhancing the formation of mRNA ribosome complexes. Background Art

[0002] Recombinant proteins are exogenous proteins expressed in host cells through genetic engineering technology and have become core tools in the field of biotechnology. Their applications are particularly prominent in the medical field, including the production of monoclonal antibody drugs, vaccine research and development, gene therapy vectors, and disease diagnostic reagents. The industrial field covers enzyme catalysis (such as cellulase, lipase), biosynthesis, and environmental degradation. In addition, recombinant proteins are used as molecular probes and signal pathway research models in basic research, promoting the development of life sciences. With the rise of precision medicine and synthetic biology, the demand for highly efficient and highly active recombinant proteins has increased sharply, and their expression efficiency directly determines the R & D cost and industrialization feasibility.

[0003] Currently, the strategies for increasing the expression level of recombinant proteins mainly focus on optimizing the host system and regulating gene expression. At the vector design level, the use of strong promoters (such as T7, pLac), multi-copy plasmids, and codon preference optimization can enhance transcription and translation efficiency; fusion tags (such as His-Tag, SUMO) can improve protein solubility and simplify the purification process. In terms of host engineering, knocking out protease genes (such as the lon / tig mutants of Escherichia coli) or co-expressing molecular chaperones (such as GroEL-GroES) can reduce protein degradation and promote correct folding. Optimization of culture conditions involves fine-tuning of induction temperature, pH value, carbon source supply, and inducer concentration (such as IPTG gradient regulation). In recent years, metabolic engineering strategies (such as regulating energy metabolic pathways) and CRISPR-mediated dynamic gene expression regulation technologies have also shown potential.

[0004] Although the existing methods have achieved certain results, how to further increase the expression level of the target protein on the existing basis remains a problem worthy of exploration. Summary of the Invention

[0005] Currently, the T7 expression system is widely used for expressing recombinant proteins in Escherichia coli, such as plasmids of the pET series (pET-21, pET-28, pET-Duet-1, etc.). Its expression cassette mainly includes a T7 promoter, a multiple cloning site (for inserting the sequence of the target protein), and a T7 terminator. The strength of the promoter in the expression cassette directly determines the transcription level of the mRNA of the target protein, and the transcription level of the mRNA is directly related to the expression level of the target protein. The T7 promoter is the strongest known prokaryotic promoter and is initiated by T7 RNA polymerase (T7 RNAP).

[0006] In prokaryotes, transcription and translation are almost simultaneous and occur in close proximity, enabling direct coordination and cooperation between RNA polymerase (RNAP) and ribosomes. When ribosomal protein bS1 is present in the small subunit (30S) of the ribosome, nascent mRNA can bind to it more efficiently. The bS1 protein helps unwind the mRNA in preparation for translation inside the ribosome (Webster, Michael W., et al. "Molecular basis of mRNA delivery to the bacterial ribosome." Science 386.6725 (2024): eado8476.). Therefore, we hypothesized that co-expressing the bS1 protein while expressing the target protein could form a bridge between RNAP and ribosomes, enabling the target mRNA to bind to ribosomes more efficiently and increasing its translation efficiency and the expression level of the target protein. If this hypothesis holds true, further increasing the expression level of the target protein would still be possible even when using the T7 expression system with the strongest promoter capacity, which would have great promotional significance.

[0007] However, experiments found that inducing the expression of the bS1 protein and the target protein simultaneously did not increase the expression of the target protein. After optimizing the expression time and the expression level of the bS1 protein, it was found that when the expression of the target protein was induced first, followed by a time interval and then the induction of the bS1 protein expression, and the bS1 expression level was controlled at a certain level, the expression level of the target protein would increase significantly. In summary, we discovered a method to further increase the expression level of the target protein when using the T7 expression system by co-expressing ribosomal bS1 protein.

[0008] The present invention provides the following technical solutions: The present invention first provides a prokaryotic expression vector or combination, which includes a first expression element of a target protein gene controlled by a first inducible promoter, and a second expression element of a ribosomal bS1 protein gene controlled by a second inducible promoter; The first and second expression elements are constructed on different vectors or on the same vector; and the first and second inducible promoters are different.

[0009] Specifically, the first inducible promoter is an IPTG-inducible promoter; the second inducible promoter is an arabinose-inducible promoter.

[0010] More specifically, the first expression element is constructed on a pET plasmid, such as the pET28a(+) plasmid, and the second expression element is constructed on a pBAD plasmid.

[0011] Among them, the gene of ribosomal protein bS1 or any gene encoding this functional protein can be used, and its coding sequence can be these specific gene sequences and their degenerate sequences.

[0012] The present invention provides a recombinant bacterium containing the prokaryotic expression vector or combination, preferably Escherichia coli.

[0013] The present invention further provides a method for increasing the protein expression level by enhancing the formation of mRNA-ribosome complexes, which includes the following steps: S1 Culturing the recombinant bacterium; S2 First inducing the expression of the target protein; S3 Then inducing the expression of ribosomal bS1 protein.

[0014] Specifically, in S2, when the recombinant bacterium is cultured to OD 600 from 0.6 to 0.8, the expression of the target protein is induced.

[0015] Preferably, in S3, when the recombinant bacterium is cultured to OD 600 from 1.0 to 1.2, the expression of low-concentration bS1 protein is induced, so as to achieve the purpose of increasing the expression level of the target protein.

[0016] In a specific embodiment, when the second expression element is constructed on the pBAD vector, 0.04%-0.06% arabinose is used for induction.

[0017] The present invention proposes a unique method that can be used to improve the expression of target proteins through research, which has broad application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of ribosomal protein bS1 promoting the efficient binding of nascent mRNA and ribosomes.

[0019] Figure 2 Maps of pET28a(+) plasmid and pBAD plasmid.

[0020] Figure 3 Effect of simultaneous induction of expression of target protein and bS1 protein on the expression level of target protein. Lane 2, 0.4 M IPTG; Lane 3, 0.4 M IPTG + 0.05% arabinose induction; Lane 4, 0.4 M IPTG + 0.2% arabinose induction.

[0021] Figure 4Effect of the induced expression time of bS1 protein on the expression level of the target protein. A) SDS-PAGE electrophoresis of the target protein. Lane 1: induced with 0.4 M IPTG + 0.2% arabinose; Lane 2: induced with 0.4 M IPTG; Lane 3: induced with 0.4 M IPTG + 0.05% arabinose. B) Fluorescent characterization of the target protein expression. Detailed implementation manners

[0022] The present invention will be further described below through specific examples for better understanding, but it does not constitute a limitation to the present invention.

[0023] Based on the research of the present invention, the principle that ribosomal protein bS1 promotes the efficient binding of nascent mRNA and ribosomes is as Figure 1 shown. When DNA is transcribed, after RNA polymerase binds to the promoter, transcription is initiated and mRNA is synthesized. At this time, bS1 protein can promote the binding of the newly transcribed mRNA by ribosomes and start translation. When the bS1 protein is insufficient, ribosomes need to recognize the SD sequence and pair with the newly transcribed mRNA to form a binding and then initiate translation, and its efficiency is lower than that when the bS1 protein participates.

[0024] Based on this principle, the following experimental studies are carried out in the present invention.

[0025] Example 1: Construction of expression plasmids Construction of plasmids pET-28a-Nanoluciferase and pBAD-bS1: Insert the gene sequence of the synthesized protein Nanoluciferase (GenBank: AFI79291.1) into the pET-28a(+) plasmid ( Figure 2 ), between NcoI and BamHI, to obtain plasmid pET-28a-Nanoluciferase; insert the gene sequence of the synthesized ribosomal protein bS1 (NCBI Gene ID: 945536) into the pBAD plasmid ( Figure 2 ), between NdeI and XhoI, to obtain plasmid pBAD-bS1.

[0026] Example 2: Effect of co-inducing the expression of the target protein and bS1 protein on the expression level of the target protein 1) Transfer the plasmids in Example 1 into Escherichia coli BL21(DE3) at the same time, screen to obtain positive transformants, inoculate them into LB medium containing 50 mg / L kanamycin and 100 mg / L ampicillin, and culture overnight at 37°C to obtain a seed solution; 2) Inoculate the seed solution into TB medium containing 50 mg / L kanamycin and 100 mg / L ampicillin at an inoculum size of 1%, and culture at 37°C until OD 600 reaches 0.6 - 0.8. Meanwhile, add 0.4 mM IPTG and 2 g / L (or 0.5 g / L) arabinose to induce the expression of the target protein and bS1 protein, and culture at 37°C for 4 - 6 h. Then collect the cells.

[0027] 3) Measure OD 600 using a spectrophotometer. Take the same OD 600 amount of bacterial cells, centrifuge at 12,000 rpm for 1 min, discard the supernatant, resuspend the bacterial cells with PBS and adjust OD 600 to 20. Take 80 µL of the sample and add 20 µL of 5×SDS protein loading buffer, and perform denaturation treatment at 100°C for 10 min. Centrifuge at 12,000 rpm for 1 min, and take 10 µL of the supernatant to run SDS - PAGE to detect the expression of the target protein and bS1.

[0028] 4) Select the endogenous protein as the internal reference, and use Image J to estimate the relative gray values of the internal reference protein and the target protein (target protein gray value / internal reference protein gray value) respectively. Take the expression level of the target protein when bS1 protein is not induced (control) as 1, and the ratio of the relative gray value of the target protein at different inducer (arabinose) concentrations to that of the control is the relative expression level of the target protein at different bS1 protein expression levels.

[0029] As Figure 3 can be seen, when the target protein (Nanoluciferase) and bS1 protein are induced to express simultaneously: low - expression bS1 has little effect on the expression level of the target protein, while high - expression of bS1 protein will lead to an increase in the expression of impurity proteins and a decrease in the expression of the target protein (Table 1).

[0030] Table 1

[0031] Example 3: Effect of the induction expression time of bS1 protein on the expression level of the target protein 1) Transfer the plasmids in Example 1 into Escherichia coli BL21(DE3) simultaneously, screen for positive transformants and inoculate them into LB medium containing 50 mg / L kanamycin and 100 mg / L ampicillin, and culture overnight at 37°C to obtain the seed solution; 2) Inoculate the seed solution into TB medium containing 50 mg / L kanamycin and 100 mg / L ampicillin at an inoculum size of 1%, and culture at 37°C until OD 600To 0.6 - 0.8, add 0.4 mM IPTG to induce the expression of the target protein; wait until OD 600 reaches 1.0 - 1.2, add 0.5 g / L of L - arabinose to induce the expression of bS1 protein, continue culturing at 37 °C for 2 - 4 h, and collect the cells.

[0032] 3) Measure OD with a spectrophotometer 600 , take the same OD 600 amount of bacterial cells, centrifuge at 12000 rpm for 1 min, discard the supernatant, resuspend the bacterial cells with PBS and adjust OD 600 to 20. Take 80 µL of the sample, add 20 µl of 5×SDS protein loading buffer, and perform denaturation treatment at 100 °C for 10 min. Centrifuge at 12000 rpm for 1 min, take 10 uL of the supernatant and run SDS - PAGE to detect the expression of the target protein and bS1.

[0033] 4) Resuspend the bacterial cells with PBS and adjust OD 600 to 20. Take an appropriate amount of the bacterial solution and add it to a 96 - well plate, add an appropriate amount of the substrate Furimazine, and measure the fluorescence intensity of Nanoluciferase with an enzyme - linked immunosorbent assay (ELISA) reader to further quantify the expression of the target protein.

[0034] When OD 600 reaches 0.6 - 0.8, first induce the expression of the target protein (Nanoluciferase). When OD 600 reaches 1.0 - 1.2 and induce the expression of bS1 protein: low - expression bS1 protein (0.4 mM IPTG + 0.05% arabinose) promotes the expression of the target protein; while high - expression of bS1 protein reduces the expression of the target protein ( Figure 4 in A). The excitation light and emission light are 400 nm and 460 nm respectively to measure the absorption light intensity of Nanoluciferase. The results Figure 4 are shown in B. When induced with 0.4 mM IPTG + 0.05% arabinose, the fluorescence intensity of Nanoluciferase increases by 1.91 - fold (Table 2).

[0035] Table 2

[0036] In summary, when using the widely used pET expression system (T7 promoter), when OD 600 reaches 0.6 - 0.8, first induce the expression of the target protein. When OD 600 reaches 1.0 - 1.2, inducing the expression of low - concentration bS1 protein (when choosing the pBAD vector, induce with 0.05% arabinose) can significantly increase the expression level of the target protein.

Claims

1. A prokaryotic expression vector or combination, characterized in that, It includes a first expression element of a target protein gene controlled by a first inducible promoter, and a second expression element of a ribosomal bS1 protein gene controlled by a second inducible promoter; The first and second expression elements are constructed on different vectors, or on the same vector; and the first and second inducible promoters are different.

2. The prokaryotic expression vector or combination according to claim 1, characterized in that: The first inducible promoter is an IPTG inducible promoter; the second inducible promoter is an arabinose inducible promoter.

3. The prokaryotic expression vector or combination according to claim 1, characterized in that: The first expression element is constructed on a pET plasmid, such as a pET28a(+) plasmid, and the second expression element is constructed on a pBAD plasmid.

4. A recombinant bacterium containing the prokaryotic expression vector or combination as claimed in any one of claims 1 to 3, preferably, it is Escherichia coli.

5. A method for increasing protein expression by enhancing the formation of mRNA ribosome complexes, characterized in that: The steps include: S1. cultivating the recombinant bacteria as claimed in claim 4; S2 first induces the expression of the target protein; S3 further induces the expression of ribosomal bS1 protein.

6. The method according to claim 5, characterized in that S2 is the culture of the recombinant bacteria to OD 600 When the concentration reaches 0.6~0.8, the expression of target protein is induced.

7. The method according to claim 5, characterized in that S3 is the culture medium for the recombinant bacteria until OD 600 When the concentration of bS1 protein reaches 1.0-1.2, it induces the expression of low concentration bS1 protein, thereby achieving the purpose of increasing the expression level of the target protein.

8. The method according to claim 7, characterized in that When the second expression element was constructed in the pBAD vector, it was induced with 0.04%-0.06% arabinose.

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