Method for improving protein biosynthesis efficiency according to tRNA expression characteristics

Abstract

The invention belongs to the technical field of biotechnology, and particularly relates to a method for improving protein biosynthesis efficiency according to tRNA expression characteristics. The invention provides a method for improving protein biosynthesis efficiency according to tRNA expression characteristics. The method comprises the following steps: (1) constructing a tRNA-seq library of a sample; (2) selecting codons corresponding to isostatic receptor tRNA with the highest expression quantity in the same homotype tRNA as high RSAU codons; and (3) replacing the low-RSAU codon in the gene sequence to be expressed with the high-RSAU codon. The method has the beneficial effects that (1) the codon preference is optimized, the use efficiency of the isoworking tRNA is improved, the concentration of the codon and the tRNA is balanced, and the translation efficiency is improved; (2) the expression quantities of isoworking tRNA of different hosts are considered, so that codons are more suitable for host cell environments; and (3) rare codons are replaced, tRNA abundance is also considered, and more comprehensive codon optimization is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology, and in particular relates to a method for improving protein biosynthesis efficiency according to tRNA expression characteristics. Background Art

[0002] Protein is the main functional molecule responsible for life activities, and its synthesis process is called translation. This process uses mRNA as a template and, with the participation of ribosomes, converts the nucleotide sequence on mRNA into the corresponding amino acid sequence through tRNA to synthesize a polypeptide chain. Protein translation is the basis for the growth, development and maintenance of life activities of organisms. Its translation efficiency is one of the important means for cells to regulate gene expression and is regulated by many factors. In the process of protein translation, the use of codons plays multiple roles in regulating gene expression and protein structure through translation-dependent and translation-independent mechanisms. The efficiency of synonymous codons recognized by cognate tRNA is different. In different eukaryotes and prokaryotes, codon usage deviation is related to the level of cognate tRNA or the number of tRNA gene copies (DOI:10.1186 / s12964-020-00642-6). Therefore, the choice of codons affects translation efficiency.

[0003] It has been determined that codons play an important role in regulating the translation rate during the elongation of mRNA translation (DOI:10.1146 / annurev-biochem-052621-091808). Generally, the use of host-preferred codons can effectively improve the translation rate and efficiency of ribosomes, thereby reducing the stalling phenomenon during the translation process; in contrast, the use of rare codons may cause the stalling of ribosomes on mRNA, which may cause premature termination of translation (DOI:10.1126 / science.aad9868). Codon optimality affects gene expression at the level of translation efficiency. The difference between reporters enriched with optimal codons and non-optimal codons at the protein level is often greater than that at the RNA level (DOI:10.1038 / s41598-020-74091-z). Therefore, codon optimization can increase the translation efficiency of endogenous and exogenous proteins in a variety of eukaryotic and prokaryotic organisms, significantly improve the stability of mRNA and the efficient and large-scale expression of proteins.

[0004] The basic principle of codon optimization is to replace rare codons with synonymous codons with high frequency of use in host cells to adapt to the preferences of host cells, thereby optimizing the translation process. In fact, a large number of studies have shown that the relative abundance and availability of tRNA are the rate-limiting steps of translation efficiency and have an impact on the stability dynamics mediated by codons. The expression of tRNA is tissue-specific, and the regulatory characteristics of any given codon may vary according to the conditions of tRNA availability and tissue-specific differences (72-74). Therefore, the relative abundance of tRNA is an important factor for codon optimization. The traditional codon optimization method is to use the Codon Adaptation Index (CAI) as an important indicator, while considering factors such as mRNA secondary structure and GC content to optimize the codon sequence of the target gene. There is no excessive consideration of the abundance changes of tRNA in different tissues and cells. For this reason, the present invention proposes an optimization method for improving protein biosynthesis efficiency according to tRNA expression characteristics.

[0005] Codon optimization refers to improving the translation efficiency of proteins by changing the codons in the mRNA sequence to make it more suitable for the codon usage preference of the host cell. The traditional codon optimization strategy is to replace the rare codons in the exogenous mRNA sequence with synonymous codons that are frequently used in the host cell, and to generate different mRNA sequences by using a series of quantitative methods to reduce the time it takes for the ribosome to find a matching tRNA on the mRNA, thereby increasing the translation rate. The approach is to optimize the codon sequence of the target gene by calculating the Codon Adaptation Index (CAI) of the original amino acid sequence in the host cell as the main indicator and taking into account optimization indicators such as mRNA secondary structure and GC content, in order to improve its speed and efficiency in the translation process. Although the traditional codon optimization strategy is simple and easy to implement, it has certain defects and shortcomings:

[0006] (1) Traditional methods usually use CAI as the main indicator for codon optimization and only replace rare codons. They cannot fully consider multiple factors such as codon usage frequency, mRNA structure, and tRNA abundance, so the optimization effect is limited;

[0007] (2) The optimized sequence contains host-biased codons, so the transcribed mRNA contains a higher proportion of codon subsets, which leads to an imbalance of different tRNAs, ultimately leading to tRNA consumption and translation termination;

[0008] (3) Traditional methods do not take into account the different codon preferences of different host cells, and the optimization method lacks universality. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0010] The present invention provides a method for improving protein biosynthesis efficiency according to tRNA expression characteristics, the method comprising the following steps:

[0011] (1) Constructing a tRNA-seq library of the sample;

[0012] (2) Data analysis;

[0013] The proportion of tRNAs transporting the same type of amino acids was calculated, and the codon corresponding to the isoacceptor tRNA with the highest expression level among the same type of tRNA was defined as the high RSAU codon;

[0014] (3) Codon optimization

[0015] Replace low RSAU codons in the gene sequence to be expressed with high RSAU codons.

[0016] Preferably, the construction of the tRNA-seq library in step (1) specifically includes the separation, pretreatment, double linker ligation of tRNA, and the synthesis and library amplification of cDNA.

[0017] Preferably, the calculation formula of RSAU in step (2) is as follows:

[0018]

[0019] Preferably, in step (1), the sample is human embryonic kidney cell line 293T.

[0020] Preferably, in step (2), the high RSAU codons are GCT, AGG, AAC, GAC, TGC, GAG, CAG, GGC, CAC, ATT, CTG, AAG, ATG, TTC, CCT, AGC, ACA, TGG, TAC, and GTG, respectively.

[0021] Beneficial effects of the present invention:

[0022] (1) Optimize codon preference, improve the efficiency of isomeric tRNA usage, balance codon and tRNA concentrations, and improve translation efficiency;

[0023] (2) Consider the expression levels of iso-tRNAs of different hosts to make the codons more adaptable to the host cell environment;

[0024] (3) Not only rare codons are replaced, but also tRNA abundance is considered to achieve more comprehensive codon optimization. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The overall flow chart for tRNA-seq library construction;

[0026] Figure 2 It is the density map of cells at the time of transfection;

[0027] Figure 3 are the results of codon optimization; A. relative expression of luciferase reporter gene after codon optimization; B. expression of luciferase reporter gene at mRNA level after codon optimization; C. translation efficiency of luciferase reporter gene after codon optimization. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are 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.

[0029] The present invention selects human embryonic kidney cell line 293T as a host, uses psiCHECK-2 plasmid as a vector, and performs sequence modification on the Firefly luciferase gene sequence, and utilizes the characteristic that luciferase combines with a substrate to generate a chemiluminescent reaction, so as to construct a luciferase reporter plasmid. By analyzing the full-length sequencing of tRNA in the cell and calculating the relative synonymous codon usage (RSAU) according to the expression abundance of the isoacceptor tRNA, the codon with the highest RSAU corresponding to each codon family is selected, and the codon corresponding to the highest RSAU (i.e., high tRNA abundance) (i.e., high RSAU codon in corresponding Table 1) replaces the original Firefly luciferase gene sequence. It should be emphasized that in the process of codon optimization, we set up three experimental groups, namely, the psiCHECK-2-cell group in which the Firefly luciferase gene sequence was directly replaced with the highest RSAU codon; the psiCHECK-2-RSAU group in which the Firefly luciferase gene sequence was subjected to traditional codon optimization and then replaced with the highest RSAU codon; and the psiCHECK-2-optimal group in which the Firefly luciferase gene sequence was simply subjected to traditional codon optimization. Subsequently, the optimized protein level and translation efficiency were detected and analyzed and compared using dual luciferase reporter gene experiments and qRT-PCR experiments, and the experimental results fully confirmed the feasibility and effectiveness of the method for improving protein biosynthesis efficiency according to tRNA expression characteristics in the present invention.

[0030] The specific experimental steps are as follows:

[0031] 1. Construction of tRNA-seq library

[0032] Human embryonic kidney cell line 293T was selected as the sample. Figure 1 Construct a tRNA-seq library using the library construction process in .

[0033] From top to bottom, they are tRNA isolation, pretreatment, double adapter ligation, cDNA synthesis and library amplification.

[0034] 2. Data Analysis

[0035] After the preprocessing of the sequencing data was completed, the software package "mim-tRNAseq" was used to perform a series of analyses on the tRNA data. The proportion of isotype tRNA was calculated. We defined the codons corresponding to the isoacceptor tRNA with the highest expression level in the same isotype tRNA as high RSAU codons, and the rest as low RSAU codons. The RSAU calculation formula is as follows:

[0036]

[0037] The high RSAU codon sequences corresponding to the codons of the twenty amino acids calculated are shown in Table 1 below:

[0038] Table 1 Codons corresponding to twenty amino acids and high RSAU codon sequences

[0039]

[0040]

[0041]

[0042] 3. Codon Optimization

[0043] The high RSAU codon calculated in the previous step was used to replace the Fireflyluciferase gene sequence of the original psiCHECK-2 plasmid. Three experimental groups were set up, namely the psiCHECK-2-cell group in which the Firefly luciferase gene sequence was directly replaced with the highest RSAU codon; the psiCHECK-2-RSAU group in which the Firefly luciferase gene sequence was optimized by traditional codons and then replaced with the highest RSAU codon; and the psiCHECK-2-optimal group in which the Fireflyluciferase gene sequence was simply optimized by traditional codons. At the same time, the wild-type psiCHECK-2 plasmid was used as the control group. The optimized Firefly luciferase gene sequence was designed and the plasmid was constructed by General Biotech.

[0044] 4. Dual luciferase reporter gene assay

[0045] 1. Cell plating (293T cells)

[0046] 12-well plate was plated, with each well being plated at 1 / 45 of the bottom area of ​​a 10 cm dish. The cell density during transfection was Figure 2 As shown:

[0047] 2. Transfected cells (taking one well of a 12-well plate as an example)

[0048] (1) Use 50ul DMEM medium (without serum and double antibody) to dilute 1ug plasmid and mix gently.

[0049] (2) Before use, gently mix Lipo2000, and then dilute 2 μl in 50 μl DMEM medium (without serum and double antibody).

[0050] (3) Mix (1) and (2) (total volume 100 μl), mix gently and incubate at room temperature for 15 min.

[0051] (4) During the incubation period, remove the well plate from the incubator and replace it with fresh culture medium without dual antibody.

[0052] (5) After incubation, gently add 100 μl of the complex to the well containing the cells to be transfected and gently shake the plate to mix.

[0053] (6) After 6 hours of culture, observe the cell status. If the cells are in good condition, there is no need to change the medium.

[0054] (7) After 48 hours, cells were harvested for subsequent experiments.

[0055] 3. Dual luciferase reporter gene experiment (the reagents used in the experiment must return to room temperature)

[0056] (1) Aspirate the cell culture medium, wash twice with PBS, add 150 μl Cell Lysis Buffer (1×) to each well, and lyse at room temperature for 20 min, pipetting up and down every 5 min.

[0057] (2) Pipette the lysate into a 1.5 ml centrifuge tube and centrifuge at 12,000 rpm and 4°C for 30 min. Take the supernatant and transfer it to a new centrifuge tube. It can be frozen and stored in a -20°C refrigerator for later use.

[0058] (3) Firefly luciferase reaction detection

[0059] Add 50 μl of Luciferase Substrate balanced at room temperature to the ELISA plate, carefully pipette 20 μl of cell lysis supernatant into the ELISA plate, quickly mix well, and immediately detect the activity of the Firefly luciferase reporter gene in an ELISA reader.

[0060] (4) Renilla Luciferase reaction detection

[0061] Add 100 μl of freshly prepared Renilla Substrate working solution to the above reaction solution, mix quickly and immediately detect the Renilla Luciferase reporter gene activity in an ELISA reader.

[0062] 5. Fluorescence quantitative PCR

[0063] 1. Trizol method to extract RNA

[0064] (1) Take 60ul of the remaining cell lysate from the dual luciferase assay, add 1ml of Trizol, mix well, and place the homogenate sample at room temperature for 5min to completely separate the nucleic acid-protein complex.

[0065] (2) According to the instructions, add 0.2 mL of chloroform to every 1 mL of TRNzol Universal reagent, cover the centrifuge tube, mix by manual inversion for 2 min, and let it stand at room temperature for 5 min.

[0066] (3) Set the centrifuge conditions to 4°C, 12,000 rpm, and 20 min. Gently place the sample in the centrifuge for centrifugation. After centrifugation, the sample will be divided into three layers, from bottom to top: pink organic phase, white middle layer, and colorless upper aqueous phase. RNA is mainly present in the upper aqueous phase. Transfer the aqueous phase to a new enzyme-free 1.5 ml centrifuge tube (about 600 ul) and mark it.

[0067] (4) Add isopropanol in an equal volume to the aqueous phase, mix thoroughly by inverting, and leave at room temperature for 10 min.

[0068] (5) Set the centrifuge conditions to 4°C, 12,000 rpm, and 10 min. Gently place the mixed centrifuge tube into the centrifuge for centrifugation. After centrifugation, remove the supernatant. The gelatinous precipitate formed on the side and bottom of the centrifuge tube is RNA.

[0069] (6) Add 1 mL of 75% ethanol (prepared with RNase-free ddH2O) to the centrifuge tube containing the RNA precipitate to wash the precipitate.

[0070] (7) Set the centrifuge conditions to 4°C, 10,000 rpm, and 5 min. Place the centrifuge tube from the previous step into the centrifuge tube and centrifuge. After centrifugation, pour out the liquid, but be careful not to pour out the precipitate. Repeat steps (7) and (8) once. After pouring out the liquid for the last time, perform a slow centrifugation and use a pipette to suck out all the liquid in the centrifuge tube. Be careful not to discard the precipitate.

[0071] (8) Open the lid of the centrifuge tube and let it dry at room temperature for 5 min. Then, according to the experimental needs, add RNase-free ddH2O to the centrifuge tube to dissolve the RNA (this time, only 12 μl was added due to concerns that the amount of RNA would be relatively small). Repeatedly pipette and mix to fully dissolve the RNA.

[0072] (9) Finally, the NanoDrop instrument was used to detect RNA concentration and purity. The results are as follows

[0073] 2. Reverse transcription of RNA into cDNA

[0074] (1) Genomic DNA removal

[0075] Prepare the following mixture in an RNase-free centrifuge tube:

[0076]

[0077] Mix gently by pipetting and incubate at 42°C for 2 min.

[0078] (2) Preparation of first-strand cDNA synthesis reaction solution

[0079]

[0080] (3) PCR instrument for first-strand cDNA synthesis reaction

[0081]

[0082] 3. Fluorescence quantitative PCR (qRT-PCR)

[0083] (1) Before the experiment, prepare the template cDNA sample, required primers (10 μm) and qRT-PCR kit. Thaw on ice, vortex and mix, centrifuge and place on ice for later use. The primer sequences are as follows:

[0084]

[0085]

[0086] (2) Prepare the reaction system, 10ul / well, as follows:

[0087]

[0088] (3) Set up the reaction program on the Bio-Rad CFX96 qRT-PCR instrument and select the two-step amplification program:

[0089] 95℃1min→95℃20s, 60℃45s(39cycles)

[0090] (4) After the reaction, the CT values ​​of luciferase and Renilla were derived and read, with Rluc as the internal reference and 2 -ΔΔCt To calculate the relative expression level of luciferase.

[0091] 6. Statistical Analysis

[0092] By comparing the ratio of the dual luciferase reporter gene to the 2 -ΔΔCt The translation efficiency was obtained by comparison. All data of dual luciferase reporter gene experiment and fluorescence quantitative PCR were statistically analyzed using GraphPad Prism 8.0 software. Each experiment was repeated four times according to biological standards, and the results were presented as mean ± standard error of the mean (SEM). Student's t-test was used for analysis and all data were standardized and marked * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.001 indicated that the difference was statistically significant.

[0093] The Firefly luciferase gene sequence of the psiCHECK-2 plasmid was codon optimized by using this method, the traditional method, and a combination of the two methods, and the wild-type psiCHECK-2 plasmid was used as a control. The above plasmids were respectively transformed into 293T cells, and then the optimized protein expression level and translation efficiency were detected and analyzed by dual luciferase reporter gene experiment and qRT-PCR experiment. The results are as follows: Figure 3 As shown in AC, from Figure 3 It can be seen from C that the translation efficiency is significantly increased after codon optimization by this method.

Claims

1. A method for improving protein biosynthesis efficiency based on tRNA expression characteristics, characterized in that: The method comprises the following steps: (1) Construction of sample tRNA-seq library (2) Data Analysis The proportion of tRNAs transporting the same type of amino acids was calculated, and the codon corresponding to the isoacceptor tRNA with the highest expression level among the same type of tRNA was defined as the high RSAU codon; (3) Codon optimization The low RSAU codons in the gene sequence to be expressed are replaced with high RSAU codons.

2. The method according to claim 1, characterized in that In step (1), constructing a tRNA-seq library specifically includes tRNA separation, pretreatment, double linker ligation, cDNA synthesis, and library amplification.

3. The method according to claim 1, characterized in that The calculation formula of RSAU in step (2) is as follows:

4. The method according to claim 1, characterized in that: In step (1), the sample is the human embryonic kidney cell line 293T.

5. The method according to claim 1, characterized in that In step (2), the high RSAU codons are GCT, AGG, AAC, GAC, TGC, GAG, CAG, GGC, CAC, ATT, CTG, AAG, ATG, TTC, CCT, AGC, ACA, TGG, TAC, and GTG, respectively.

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