Method for improving protein expression quantity

Abstract

The invention discloses a method for improving protein expression quantity, which is characterized in that an ATG sequence of non-coding methionine in a CDS sequence is directly subjected to synonymous mutation, and then the expression quantity of target protein is obviously improved through expression, so that the production efficiency is improved, and the cost is reduced; the method does not need a bioinformatics platform to optimize codons of specific species, and is simple and efficient; the method is suitable for most CDS sequences and various host cell systems, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a method for increasing protein expression. Background Art

[0002] In genetic engineering, increasing the expression of target proteins is the key to achieving efficient bioproduction. In the biotechnology and biopharmaceutical industries, efficient expression of recombinant proteins is the key to producing drugs, vaccines and other biological products. Traditional protein expression systems, such as Escherichia coli, yeast, insect and mammalian cells, often face the problem of insufficient expression. Especially in plant systems, although they have the advantages of cost-effectiveness and scalability, protein expression levels are usually low. Traditional protein expression strategies often rely on strong promoters, codon optimization, optimized culture conditions, etc. However, these methods may sometimes not be sufficient to meet the needs of high expression levels, especially when producing recombinant vaccines or biopharmaceuticals.

[0003] Therefore, developing new strategies to increase protein expression is an important need in this field. Summary of the invention

[0004] The present invention aims to provide a method for increasing protein expression by optimizing non-coding sequences to meet the demand for increasing protein expression.

[0005] In view of this, the solution of the present invention is:

[0006] A method for increasing protein expression, comprising the steps of:

[0007] Obtain the ATG non-coding sequence consisting of any two adjacent codons in the gene coding region;

[0008] The bases of the ATG non-coding sequence are mutated to cause synonymous mutations in the corresponding codons;

[0009] The mutated coding sequence is expressed as an exogenous protein to obtain a high expression level of protein.

[0010] Furthermore, the mutation process mutates the base A or T in the ATG non-coding sequence, causing a synonymous mutation in the corresponding codon.

[0011] Furthermore, the ATG non-coding sequence contains multiple sites, and the mutation process performs synonymous mutations on more than 50% of the ATG non-coding sequences, that is, mutates more than half of the ATG non-coding sequences to form synonymous mutations corresponding to multiple codons.

[0012] Preferably, the mutation process performs synonymous mutations on all ATG non-coding sequences.

[0013] Furthermore, the protein is plant protein, animal protein or microbial protein.

[0014] Furthermore, the protein is luciferase or Basta.

[0015] Preferably, the coding sequence of luciferase is shown in SEQ ID NO: 1 or 5, and the coding sequence of luciferase after mutation is shown in SEQ ID NO: 2 or 6; the coding sequence of Basta is shown in SEQ ID NO: 3, and the coding sequence of Basta after mutation is shown in SEQ ID NO: 4.

[0016] Furthermore, the exogenous expression process constructs an expression vector based on molecular cloning, which is then transformed into a host cell culture to obtain protein expression.

[0017] Preferably, the host is one of a plant, a microorganism or an animal cell.

[0018] Preferably, the plant is a plant protoplast, selected from tobacco protoplasts, Arabidopsis protoplasts, and rice protoplasts;

[0019] Preferably, the microorganism is one of Escherichia coli and Agrobacterium;

[0020] Preferably, the animal cell is a human embryonic kidney cell.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The method of the present invention directly performs synonymous mutation of the ATG sequence of non-coding methionine in the CDS sequence, thereby significantly increasing the expression amount of the target protein through expression, which helps to improve production efficiency and reduce costs; the method does not require the use of a bioinformatics platform to perform species-specific codon optimization, is simple and efficient; is applicable to most CDS sequences and a variety of host cell systems, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of a synonymous mutation of a non-coding ATG sequence in a CDS sequence in an embodiment of the present invention.

[0024] Figure 2 Schematic diagram of vectors pSYB-Dual-FLUC and pSYB-Dual-FLUCdelATG constructed in one embodiment of the present invention.

[0025] Figure 3The comparison results of the ratios of FLUC to RLUC after the two dual luciferase vectors pSYB-Dual-FLUC and pSYB-Dual-FLUCdelATG in Example 1 of the present invention were transiently transformed into tobacco protoplasts.

[0026] Figure 4 The results are activity comparisons of the two dual luciferase vectors pSYB-Dual-FLUC and pSYB-Dual-FLUCdelATG in Example 1 of the present invention after transient transformation of Arabidopsis protoplasts.

[0027] Figure 5 The results are compared for the expression of Basta protein levels after rice protoplasts were transformed with pBWA(V)BS and Basta-delATG, respectively, in Example 1 of the present invention.

[0028] Figure 6 This is the activity comparison result after pSYB-Dual-LUC01-TBF1 and pSYB-Dual-LUC01-TBF1-LUCdelATG were expressed in Escherichia coli DH5α competent cells in Example 2 of the present invention.

[0029] Figure 7 This is the activity comparison result after pSYB-Dual-LUC01-TBF1 and pSYB-Dual-LUC01-TBF1-LUCdelATG were expressed in Agrobacterium GV3101 competent cells in Example 2 of the present invention.

[0030] Figure 8 The results are a comparison of the activities of pmirGLO and XB24-pmirGLO-FLUCdelATG after expression in 293T cells in Example 3 of the present invention. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In one embodiment, a method for optimizing the CDS sequence to increase protein expression is proposed. Without changing the protein amino acid sequence, a synonymous mutation is performed on the ATG sequence of the non-coding methionine in the CDS, thereby improving the efficiency of the translation process and thereby increasing the yield of the target protein. Specifically, the method comprises the following steps:

[0033] 1. Identify the ATG sequence in the target CDS sequence: Through base sequence analysis, determine the ATG sequence present in the CDS sequence, which includes two categories: ATG sequence encoding methionine and ATG sequence non-coding methionine. The ATG sequence non-coding methionine is composed of any two adjacent codons. Design mutation strategy: Design a synonymous mutation strategy to replace the ATG sequence non-coding methionine in the CDS sequence.

[0034] 2. Construct mutation vector: Use molecular cloning technology to construct the mutated CDS sequence expression vector.

[0035] 3. Transient transformation of protoplast cells: transform the constructed expression vectors into host cells respectively.

[0036] 4. Protein expression: Cultivate the transformed host cells to allow the expression vector to express the target protein.

[0037] In the above embodiment, by directly performing synonymous mutations on the ATG sequence of the non-coding methionine in the CDS sequence, the expression level of the target protein is significantly increased through expression, which helps to improve production efficiency and reduce costs; there is no need to use a bioinformatics platform to perform species-specific codon optimization, which is simple and efficient; and is applicable to most CDS sequences and a variety of host cell systems.

[0038] As mentioned above, synonymous mutation refers to the fact that sometimes a mutation of a base pair in a DNA fragment does not change the amino acid encoded. The reason is that the codon at this position is a degenerate codon before and after the mutation. Based on the existing synonymous mutation types, the third base in the codon is usually mutated. Therefore, for any ATG sequence of non-coding methionine composed of adjacent codons on the left and right, a synonymous mutation is performed on the codon on the left. Therefore, the mutation of the ATG sequence of non-coding methionine can be a mutation to base A or base T, and the amino acid encoded by the corresponding codon is not changed by the mutation of the base.

[0039] In some embodiments, Figure 1 Taking a synonymous mutation of a non-coding ATG sequence of the partial CDS sequence as an example, Figure 1 In A, the AT in ATG, which does not encode methionine, comes from the last two bases of TAT (encoding tyrosine, Tyr), and the G comes from the first base of GGG (encoding glycine, Gly). Figure 1 The red box in A shows the site of synonymous mutation, that is, TAT mutates to TAC, and both codons encode tyrosine. Figure 1 As shown in B, the arrows indicate the positions of synonymous mutations of ATG of non-coding methionine. Synonymous mutations were performed on 14 non-coding methionine ATGs in the CDS sequence of FLUC, and the mutated sequence was named FLUC-delATG.

[0040] In a preferred embodiment, in a CDS sequence, the ATG non-coding sequence includes multiple sites, and the mutation process performs synonymous mutation on at least one ATG non-coding sequence to increase protein expression; more preferably, multiple sites are mutated, for example, more than 50% of the ATG non-coding sequences are mutated synonymously, that is, more than half of the ATG non-coding sequences are mutated to form synonymous mutations corresponding to multiple codons. The mutated ATG non-coding sequences can be randomly distributed, such as continuous ATG non-coding sequence mutations, or ATG non-coding sequences with intervals.

[0041] In a more preferred embodiment, synonymous mutations are performed on all ATG non-coding sequences to increase the expression level of the target protein as much as possible.

[0042] In a preferred embodiment, the mutation process can accurately perform synonymous mutations on the ATG sequence in CDS based on gene editing tools such as CRISPR-Cas9, while advances in synthetic biology have made it possible to design and construct new expression vectors that can more efficiently express target proteins in host cells.

[0043] In a preferred embodiment, the protein is a plant protein, an animal protein or a microbial protein, such as luciferase or Basta. Preferably, the coding sequence of luciferase is as shown in SEQ ID NO: 1 or 5, and correspondingly, the coding sequence of luciferase after mutation is as shown in SEQ ID NO: 2 or 6; the coding sequence of Basta is as shown in SEQ ID NO: 3, and the coding sequence of Basta after mutation is as shown in SEQ ID NO: 4.

[0044] In a preferred embodiment, the protein expression can be carried out by expressing the sequence after synonymous mutation in bacteria, yeast, animal cells or plant cells. For plants, protoplasts can be selected, including but not limited to tobacco protoplasts, Arabidopsis protoplasts, and rice protoplasts; for microorganisms, including but not limited to Escherichia coli and Agrobacterium; for animal cells, human embryonic kidney cells are not limited to use.

[0045] In the above embodiment, by performing synonymous mutations on the ATG sequence of non-coding methionine in CDS, the efficiency of the translation process is improved, thereby increasing the yield of the target protein, such as tens of times or hundreds of times. The method for increasing protein expression is widely used, such as in the field of drug production, by increasing the expression of specific drug proteins, production costs can be reduced and the accessibility of drugs can be improved. In basic research, enhancing the expression of specific proteins helps to deeply understand their functions and mechanisms of action. In agricultural biotechnology, increasing the expression of specific proteins in crops may enhance the disease resistance of crops or improve nutritional value.

[0046] Example 1 Optimizing CDS sequences in plants to increase protein expression

[0047] 1. Expression of target protein in tobacco protoplasts

[0048] Based on the FLUC coding sequence shown in SEQ ID NO: 1, a synonymous mutation was performed on the ATG sequence of the non-coding methionine according to the strategy of synonymous mutation to obtain a mutated sequence as shown in SEQ ID NO: 2. The mutated sequence was named FLUC-delATG. Figure 2 As shown, the FLUC and FLUC-delATG sequences were cloned into expression vectors, and the constructed vectors were named pSYB-Dual-FLUC and pSYB-Dual-FLUCdelATG, respectively.

[0049] The pSYB-Dual-FLUC and pSYB-Dual-FLUCdelATG expression vectors were used to transiently transform tobacco protoplasts, respectively. Although FLUCdelATG mutated the ATG of the non-coding methionine at the 14th position of the CDS interval based on FLUC, the amino acid sequences encoded by the two are consistent. Therefore, the effect of different CDS sequences on translation efficiency can be detected by protease activity. In the dual luciferase system, RLUC is used as an internal reference to effectively eliminate the interference caused by different transformation efficiencies. The tobacco protoplast cells were lysed, the protein was extracted, and the activities of FLUC and RLUC were detected by a microplate reader. The difference between the experimental group (pSYB-Dual-FLUCdelATG) and the control group (pSYB-Dual-FLUC) was detected by the ratio of FLUC to RLUC, and the results are shown in the figure. Figure 3 (n=6, the figure shows the mean ± SD, and the value is a multiple relationship). As shown in the figure, the FLUC / RLUC value of the pSYB-Dual-FLUCdelATG expression vector is 15.62 times that of the control group pSYB-Dual-FLUC. The results show that by optimizing the coding region (CDS) sequence, that is, replacing the ATG sequence of the non-coding methionine, the expression of the target protein in the host cell can be improved.

[0050] 2. Arabidopsis protoplasts expressing target protein

[0051] The experimental operation was the same as in Example 1, and the transiently transformed recipient object was changed from tobacco protoplasts to Arabidopsis protoplasts. Arabidopsis protoplast cells were lysed, proteins were extracted, and FLUC and RLUC activities were detected by a microplate reader. The difference between the experimental group (pSYB-Dual-FLUCdel ATG) and the control group (pSYB-Dual-FLUC) was detected by the ratio of FLUC to RLUC. Figure 4(n=6, the figure shows the mean ± SD, and the value is a multiple relationship). As shown in Figure 1, by transient transformation of Arabidopsis protoplasts, the FLUC / RLUC value of the pSYB-Dual-FLUCdelATG expression vector was 25.12 times that of the control group pSYB-Dual-FLUC. The results are consistent with those of Example 1. In different host cells, replacing the ATG sequence of the non-coding methionine in the CDS interval can significantly improve the expression of the target protein.

[0052] 3. Expression of target protein in rice protoplasts

[0053] Different from the optimization of FLUC sequence in 1 and 2, this experiment selected Basta coding sequence for optimization, and constructed two different expression vectors pBWA(V)BS and Basta-delATG respectively. The Basta coding sequence is shown in SEQ ID NO: 3, and the corresponding vector is pBWA(V)BS. The coding sequence of Basta coding sequence after synonymous mutation according to the mutation strategy is shown in SEQ ID NO: 4, and the corresponding vector is Basta-delATG.

[0054] The constructed vectors pBWA(V)BS and Basta-delATG were transformed into rice protoplasts to express the Basta gene in rice protoplasts. The transformed protoplast cells were lysed to release the intracellular proteins. Subsequently, total protein samples were extracted from the lysed cells, and these samples were used for subsequent Western blot analysis.

[0055] First, equal amounts of total protein samples were subjected to electrophoresis separation, a step that separates proteins based on their molecular size and charge differences. The separated proteins were transferred to a membrane in preparation for subsequent antibody detection. Basta protein was detected using specific antibodies to ensure the accuracy of the detection. At the same time, Actin protein was used as an internal reference to standardize the experimental results to correct for variations in the experiment. The expression of different vectors was evaluated by comparing the signal intensity of Basta protein. The signal intensity directly reflects the amount of protein expressed by the corresponding vector. As shown in Figure 5, the experimental results show that the Basta-delATG vector expressed a higher level of Basta protein than the pBWA(V)BS vector in rice protoplasts.

[0056] Example 2 Optimizing CDS sequences in microorganisms to increase protein expression

[0057] 1. Expression of target protein in E. coli

[0058] The FLUC and FLUC-delATG sequences are the same as in Example 1, and the pSYB-Dual-LUC01-TBF1 and pSYB-Dual-LUC01-TBF1-LUCdelATG expression vectors are constructed respectively. This experiment aims to compare the expression efficiency of two dual luciferase vectors pSYB-Dual-LUC01-TBF1 and pSYB-Dual-LUC01-TBF1-LUCdelATG in Escherichia coli DH5α competent cells. The experiment first transformed the two vectors into Escherichia coli DH5α competent cells, and then lysed the cells to extract the total protein sample. The FLUC and RLUC activities in the samples were detected using an ELISA instrument, wherein FLUC was used as the main reporter gene and RLUC was used as an internal reference for standardization data. By calculating the ratio of FLUC to RLUC activity, the expression difference between the experimental group (pSYB-Dual-LUC01-TBF1-LUCdelATG) and the control group (pSYB-Dual-LUC01-TBF1) was detected. The results show Figure 6 (n=6, the figure shows the mean ± SD, and the value is the multiple relationship). As shown in Figure 5, the FLUC expression level in the experimental group was significantly higher than that in the control group, up to 503.55 times. The results showed that the pSYB-Dual-LUC01-TBF1-LUCdelAT G vector had a higher expression efficiency in E. coli. This result was attributed to the optimization of the FLUC sequence, which enhanced the expression of the FLUC gene.

[0059] 2. Expression of target protein in Agrobacterium

[0060] The operation was the same as 1. In this experiment, two dual luciferase vectors pSYB-Dual-LUC01-TBF1 and pSYB-Dual-LUC01-TBF1-LUCdelATG were transformed into Agrobacterium GV3101 competent cells. Proteins were extracted by lysing cells, and FLUC and RLUC activities were detected using a microplate reader. The expression difference between the experimental group and the control group was evaluated by the ratio of FLUC to RLUC. The results are shown in Figure 7 As shown in Figure 1 (n=6, the figure shows the mean ± SD, and the values ​​are multiples), the FLUC expression level of the pSYB-Dual-LUC01-TBF1-LU CdelATG vector was significantly higher than that of the pSYB-Dual-LUC01-TBF1 vector, up to 380.9 times. The results show that the optimization of the FLUC sequence also promotes the expression of the target protein in Agrobacterium.

[0061] Example 3 Optimizing CDS sequences in animal cells to increase protein expression

[0062] The coding sequence of FLUC-animal is shown in SEQ ID NO: 5. Correspondingly, the coding sequence of FLUC-animal after mutation is shown in SEQ ID NO: 6, which is recorded as FLUC-animal-delATG. In this experiment, the expression effects of two dual luciferase vectors, pmirGLO and XB24-pmirGLO-FLUCdelATG, in 293T cells (human embryonic kidney cells) were investigated. The experimental steps include transforming the two vectors into 293T cells respectively, and then lysing the cells to extract protein samples. Using a microplate reader, we detected the activity of FLUC-animal and RLUC-animal, and calculated the ratio of FLUC-animal to RLUC-animal to evaluate the expression difference between the experimental group (XB24-pmirGLO-FLUCdelATG) and the control group (pmirGLO). The results are shown in Figure 2. Figure 8 As shown in (n=3, the figure shows the mean ± SD), the experimental results show that the expression level of FLUC-animal-delATG in the experimental group was significantly higher than that in the control group. The results show that the optimization of the FLUC-animal sequence also has the effect of promoting the expression of the target protein in human embryonic kidney cells.

[0063] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for increasing protein expression, characterized in that the steps include: Obtain the ATG non-coding sequence consisting of any two adjacent codons in the gene coding region; The bases of the ATG non-coding sequence are mutated to cause synonymous mutations in the corresponding codons; The mutated coding sequence is expressed as an exogenous protein to obtain a high expression level of protein.

2. The method according to claim 1, characterized in that The mutation process mutates the base A or T in the ATG non-coding sequence, causing a synonymous mutation in the corresponding codon.

3. The method according to claim 1, characterized in that The ATG non-coding sequence includes multiple sites, and the mutation process performs synonymous mutations on more than 50% of the ATG non-coding sequences.

4. The method according to claim 3, characterized in that The mutagenesis process performed synonymous mutations on all ATG non-coding sequences.

5. The method according to claim 1, characterized in that The protein is plant protein, animal protein or microbial protein.

6. The method according to claim 1, characterized in that The protein is luciferase or Basta.

7. The method according to claim 6, characterized in that The coding sequence of the luciferase after mutation is shown in SEQ ID NO: 2 or 6; the coding sequence of the Basta protein after mutation is shown in SEQ ID NO:

4.

8. The method according to claim 1, characterized in that The exogenous expression process is based on molecular cloning to construct a recombinant expression vector, which is then transformed into a host cell culture to obtain protein expression.

9. The method according to claim 8, characterized in that The host is one of a plant, a microorganism or an animal cell.

10. The method according to claim 9, characterized in that The plant is a plant protoplast, selected from tobacco protoplasts, Arabidopsis protoplasts, and rice protoplasts; And / or, the microorganism is one of Escherichia coli and Agrobacterium; And / or, the animal cell is a human embryonic kidney cell.

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