Application of two ribosomal protein coding genes in improving growth rate of prokaryotic expression bacteria

Abstract

The invention provides application of Escherichia coli 50S ribosome subunit protein coding genes rmF and rplX in the aspect of improving the growth rate of prokaryotic expression bacteria, and particularly relates to an Escherichia coli 50S ribosome subunit protein L32 coding gene rmF as shown in SEQ ID NO: 2, an Escherichia coli 50S ribosome subunit protein L24 coding gene rplX as shown in SEQ ID NO: 4 and application of coding proteins of the Escherichia coli 50S ribosome subunit protein L24 coding gene rplX. It is found that after rmF and rplX genes are transferred into a prokaryotic expression strain, the growth rate of the prokaryotic expression strain under normal conditions can be increased. The engineering strain is high in growth rate and high in expression efficiency, and can be used as an important biological resource in related gene engineering.

Description

Technical Field

[0001] The invention belongs to the biological field, and particularly relates to the application of Escherichia coli 50S ribosomal subunit protein encoding genes rpmF and rplX in improving the growth rate of prokaryotic expression bacteria. Background Art

[0002] The ribosome is an important ribonucleoprotein particle within biological cells, typically composed of a large subunit and a small subunit. It is responsible for recognizing genetic information from mRNA and translating it into amino acid sequences. Prokaryotic ribosomes are 70S ribosomes, composed of a 50S large subunit and a 30S small subunit. The large subunit contains approximately 34 proteins that play important roles in regulating peptide synthesis and maintaining ribosome stability. rpmF, the gene encoding the 50S ribosomal subunit protein L32 of Escherichia coli, is the furthest from the DNA replication origin (oriC) among all ribosomal protein genes in E. coli, and therefore has a lower copy number than genes adjacent to oriC. However, the expression level of rpmF, specifically the L32 protein, is roughly equivalent to that of ribosomal proteins other than the L7 / L12 proteins, potentially reflecting the superiority of rpmF gene expression. rplX is the gene encoding the 50S ribosomal subunit protein L24 of Escherichia coli. L24 is an important protein in the assembly of the large ribosomal subunit and may have a positive effect on gene expression in Escherichia coli.

[0003] Using E. coli as a prokaryotic host for protein expression offers advantages such as ease of operation, cost-effectiveness, and high expression levels, making it widely used in biology, medicine, industry, and agriculture. Proper modification of E. coli prokaryotic expression bacteria to increase their growth rate can not only improve expression efficiency but also reduce culture costs, thereby enhancing scientific research or industrial production. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art and optimize prokaryotic expression strains. Gene expression analysis and functional studies of the rpmF and rplX genes encoding the 50S ribosomal subunit protein of Escherichia coli were performed. The results showed that the transgenic strains obtained by transferring these genes into prokaryotic expression strains significantly increased their growth rates under normal conditions, indicating that these strains can be used to increase bacterial growth rates.

[0005] In the present invention, the rpmF gene is a gene encoding the 50S ribosomal subunit protein L32 of Escherichia coli, the nucleotide sequence of which is SEQ ID NO: 2, and the amino acid sequence of the protein encoded by it is SEQ ID NO: 1;

[0006] The rplX gene encodes the 50S ribosomal subunit protein L24 of Escherichia coli, the nucleotide sequence of which is SEQ ID NO: 4, and the amino acid sequence of the protein it encodes is SEQ ID NO: 3;

[0007] The present invention provides several recombinant expression vectors, which include an original vector and rpmF, rplX genes or their open reading frames. The original vector can be a vector commonly used in the field of gene recombination, such as a virus, a plasmid, etc. The present invention is not limited thereto. In a specific embodiment of the present invention, the original vector uses a pET28a vector plasmid, but it should be understood that the present invention can also use other plasmids, or viruses, etc.

[0008] Preferably, the original vector is a pET28a vector plasmid, and the rpmF and rplX genes of the present invention are connected to the pET28a vector plasmid through double enzyme digestion with NcoⅠ and BamHI.

[0009] The present invention also provides the use of rpmF and rplX genes, or their corresponding proteins, or their corresponding recombinant expression vectors, in increasing the growth rate of a prokaryotic expression strain. The prokaryotic expression strain can be a prokaryotic expression strain commonly used in the field of genetic recombination, and the present invention is not limited thereto. In a specific embodiment of the present invention, the prokaryotic expression strain is Escherichia coli BL21 (DE3).

[0010] Preferably, the prokaryotic expression strain is Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0011] Beneficial effects of the present invention:

[0012] The present invention conducts gene expression analysis and functional research on the rpmF and rplX genes encoding the 50S ribosomal subunit protein of Escherichia coli, and finds that the growth rate of the transgenic strains obtained after these genes are transferred into prokaryotic expression strains is significantly improved under normal conditions. When the prokaryotic expression strains carrying the rpmF and rplX genes are applied to engineered bacteria, the growth rate of the engineered bacteria is accelerated, the working efficiency of the engineered bacteria is significantly improved, the cost is reduced, etc., and the invention has good prospects in the application of microbial genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 These are the growth rate measurements of the rpmF recombinant bacteria and the control bacteria under normal conditions.

[0014] Figure 2 These are the growth rate measurements of rplX recombinant bacteria and control bacteria under normal conditions. DETAILED DESCRIPTION

[0015] The present invention will be further described below with reference to the accompanying drawings and in conjunction with specific examples for a better understanding of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or in accordance with the product instructions were followed. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products. The LB liquid culture medium used in the examples contains: 1% peptone, 1% NaCl, and 0.5% yeast powder. The percentages stated are by mass.

[0016] (1) Construction of recombinant bacteria

[0017] 1. Construction of rpmF recombinant bacteria

[0018] The nucleotide sequence of the rpmF gene was downloaded from NCBI. Based on the nucleotide sequence and seamless cloning, a pair of specific primers was designed to amplify the full-length rpmF gene from the Escherichia coli genome. The amplification procedure was as follows: pre-denaturation at 95°C for 2 min; 20 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 15 s, and extension at 72°C for 15 s; and 25 cycles of denaturation at 95°C for 15 s, annealing at 66°C for 15 s, and extension at 72°C for 15 s.

[0019] Specific primers were designed as follows:

[0020] SEQ ID NO: 5:

[0021] AACTTTAAGAAGGAGATATAATGGCCGTACAACAGAATAAAC

[0022] SEQ ID NO: 6:

[0023] GCTCGAATTCGGATCCTTACTTAGCGATGACCTTGCG

[0024] The fragment obtained by PCR was seamlessly cloned and connected with the pET28a vector plasmid double-digested with NcoⅠ and BamHI, and sequenced. The sequencing results showed that the fragment obtained above was the rpmF gene fragment mentioned in the present invention, having the nucleotide sequence of SEQ ID NO: 2.

[0025] The recombinant plasmid obtained above was transformed into the E. coli expression strain E. coli BL21 (DE3) for induced expression, thereby obtaining the rpmF recombinant bacteria pET28a-rpmF.

[0026] 2. Construction of rplX recombinant bacteria

[0027] The nucleotide sequence of the rplX gene was downloaded from NCBI. Based on the nucleotide sequence and seamless cloning, a pair of specific primers was designed to amplify the full-length rplX gene from the Escherichia coli genome. The amplification procedure was as follows: pre-denaturation at 95°C for 2 min; 20 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 15 s, and extension at 72°C for 15 s; and 25 cycles of denaturation at 95°C for 15 s, annealing at 66°C for 15 s, and extension at 72°C for 15 s.

[0028] Specific primers were designed as follows:

[0029] SEQ ID NO: 7:

[0030] AACTTTAAGAAGGAGATATAATGGCAGCGAAAATCCGTCGT

[0031] SEQ ID NO:8:

[0032] CGGAGCTCGAATTCGGATCCTTACTTGATAGTTTCGCTGTTAGACT

[0033] The fragment obtained by PCR was seamlessly cloned and connected with the pET28a vector plasmid double-digested with NcoⅠ and BamHI, and then sequenced. The sequencing results showed that the fragment obtained above was the rplX gene fragment mentioned in the present invention, having the nucleotide sequence of SEQ ID NO: 4.

[0034] The recombinant plasmid obtained above was transformed into the E. coli expression strain E. coli BL21 (DE3) for induced expression, thereby obtaining the rplX recombinant bacteria pET28a-rplX.

[0035] (2) Functional verification of genes

[0036] The recombinant bacteria obtained above and the control bacteria (E. coli BL21 (DE3) expression strain transformed with the blank pET28a vector plasmid) were activated and cultured overnight at 37°C in LB medium containing 100 μg / mL kanamycin. The next day, the cells were transferred to 50 ml of LB medium containing 100 μg / mL kanamycin and cultured for 2 h. IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.4 mM. The OD600 values ​​were then measured every 2 h (as shown in Tables 1 and 2, as shown in Tables 1 and 2). Figure 1 、 2 ), and the growth rate of each sampling time point relative to the control bacteria was calculated (as shown in Table 3), and the calculation formula was (recombinant bacteria OD600-control bacteria OD600) / control bacteria OD600×100%.

[0037] Table 1 Growth OD600 changes of pET28a-rpmF strain and control strain (Empty) (IPTG added at 2 h)

[0038]

[0039] Table 2 Growth OD600 changes of pET28a-rplX strain and control strain (Empty) (IPTG added at 2 h)

[0040]

[0041] Table 3 OD600 increase of pET28a-rpmF strain and pET28a-rplX strain relative to the control strain (Empty) at different time points.

[0042]

[0043] As shown in Table 3, compared with the control bacteria, the rpmF recombinant bacteria increased by 77.671% in 4 hours, and the rplX recombinant bacteria increased by 54.545% in 4 hours. The experimental results show that the Escherichia coli 50S ribosomal subunit proteins encoded by rpmF and rplX can enhance the growth of prokaryotic expression bacteria.

[0044] Amino acid sequence or nucleotide sequence used in the present invention

[0045] SEQ ID NO.1:

[0046] MAVQQNKPTRSKRGMRRSHDALTAVTSLSVDKTSGEKHLRHHITADGYYRGRKVIAK*

[0047] SEQ ID NO.2:

[0048] ATGGCCGTACAACAGAATAAACCAACCCGTTCCAAACGTGGCATGCGT

[0049] CGTTCCCATGACGCGCTGACCGCAGTCACCAGCCTGTCTGTAGACAAA

[0050] ACTTCTGGTGAAAAACACCTGCGTCACCACATCACTGCCGACGGTTAC

[0051] TACCGCGGCCGCAAGGTCATCGCTAAGTAA

[0052] SEQ ID NO.3:

[0053] MAAKIRRDDEVIVLTGKDKGKRGKVKNVLSSGKVIVEGINLVKKHQKPV

[0054] PALNQPGGIVEKEAAIQVSNVAIFNAATGCADRVGFRFEDGKKVRFFKSNSETIK*

[0055] SEQ ID NO.4:

[0056] ATGGCAGCGAAAATCCGTCGTGATGACGAAGTTATCGTGTTAACCGGT

[0057] AAAGATAAAGGTAAACGCGGTAAAGTTAAGAATGTCCTGTCTTCCGGC

[0058] AAGGTCATTGTTGAAGGTATCAACCTGGTTAAAACATCAGAAGCCG

[0059] GTTCCGGCCCTGAACCAACCGGGTGGCATCGTTGAAAAAGAAGCCGC

[0060] TATTCAGGTTTCCAACGTAGCAATCTTCAATGCGGCAACCGGCAAGGC

[0061] TGACCGTGTAGGCTTTAGATTCGAAGACGGTAAAAAAGTCCGTTTCTT

[0062] CAAGTCTAACAGCGAAACTATCAAGTAA

[0063] SEQ ID NO:5:

[0064] AACTTAAGAAGGAGATAATGGCCGTACAACAGAATAAC

[0065] SEQ ID NO:6:

[0066] GCTCGAATTCGGATCCTTACTTAGCGATGACCTTGCG

[0067] SEQ ID NO:7:

[0068] AACTTAAGAAGGAGATAATGGCAGCGAAAATCCGTCGT

[0069] SEQ ID NO:8:

[0070] CGGAGCTCGAATTCGGATCCTTACTTGATAGTTTCGCTGTTAGACT

[0071] The content of the present invention is not limited to the specific embodiments listed. Any equivalent transformation of the technical solution of the present invention made by ordinary technicians in this field after reading the description of the present invention is covered by the claims of the present invention.

Claims

1. A use of the Escherichia coli 50S ribosomal subunit protein L32 encoding gene rpmF with a nucleotide sequence as shown in SEQ ID NO: 2, or a protein with an amino acid sequence as shown in SEQ ID NO: 1, or a recombinant expression vector for improving the growth rate of a prokaryotic expression strain; the recombinant expression vector comprises an original vector and the Escherichia coli 50S ribosomal subunit protein L32 encoding gene or its open reading frame.

2. A use of the Escherichia coli 50S ribosomal subunit protein L24 encoding gene rplX with a nucleotide sequence as shown in SEQ ID NO: 4, or a protein with an amino acid sequence as shown in SEQ ID NO: 3, or a recombinant expression vector for improving the growth rate of a prokaryotic expression strain; the recombinant expression vector comprises an original vector and the Escherichia coli 50S ribosomal subunit protein L24 encoding gene or its open reading frame.

3. The use according to claim 1 or 2, characterized in that The original vector is a pET28a vector plasmid, and the coding genes rpmF and rplX are connected to the pET28a vector plasmid through double enzyme digestion with NcoⅠ and BamHI.

4. The use according to claim 1 or 2, characterized in that The prokaryotic expression strain is Escherichia coli BL21 (DE3).

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