CN107857801B - A signal peptide that can be used to improve secretion efficiency and its application - Google Patents

Abstract

The invention discloses a signal peptide which can be used for improving secretion efficiency and its application. The amino acid sequence of the signal peptide is shown in SEQ ID NO.1. The nucleotide sequence encoding the signal peptide is selected from any of the following sequences: (a) the nucleotide sequence shown in SEQ ID NO.2 or its complementary sequence; (b) the nuclear sequence shown in SEQ ID NO.2 The nucleotide sequence obtained by performing one or more nucleotide substitutions, deletions or additions has the same nucleotide sequence as the nucleotide sequence shown in SEQ ID NO. 2 as a signal peptide or its complementary sequence. The invention provides a signal peptide with strong secretion and expression activity, which can realize high expression of exogenous genes and provides effective elements for Bacillus subtilis to express exogenous genes.

Description

A signal peptide that can be used to improve secretion efficiency and its application

technical field

The invention belongs to the technical field of genetic engineering, and particularly relates to a signal peptide which can be used to improve secretion efficiency and its application.

Background technique

Bacillus subtilis is a gram-positive aerobic bacillus widely used in industrial enzyme production. Compared with other prokaryotes, using Bacillus subtilis as the host for protein expression has the following advantages: 1. The genetic background is clear, and the whole genome has been sequenced; 2. There is no codon preference; 3. The fermentation is simple and has no special requirements for nutrition 4. It has a set of efficient protein secretion system, and most of the proteins secreted by it have biological activity and natural conformation; 5. It is non-pathogenic and does not produce any endotoxin.

In-depth research on expression secretion system and related secretion elements in Bacillus subtilis has been carried out. Among them, the screening of signal peptides is used as a strategy to effectively improve the secretion yield of target proteins. Signal peptide is a unique amino acid sequence located at the N-terminus of newly secreted proteins and cell membrane proteins, which has a significant impact on the transmembrane secretion efficiency of proteins. The signal peptide sequence generally does not have obvious amino acid sequence conservation, and generally consists of a positively charged N domain, a hydrophobic amino acid-rich H domain, and a negatively charged C domain. Studies have shown that there is a problem of compatibility between secreted proteins and signal peptides, and the efficiency of different signal peptides leading to the secretion of the same protein is significantly different. Therefore, the study of different kinds of signal peptides of Bacillus subtilis itself is of great significance for realizing the high-efficiency expression and secretion mechanism of foreign genes.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a signal peptide that can be used to improve secretion efficiency.

Another object of the present invention is to provide a gene encoding the signal peptide that can be used to improve secretion efficiency.

Another object of the present invention is to provide the application of the signal peptide which can be used to improve secretion efficiency.

The object of the present invention is achieved through the following technical solutions: a signal peptide that can be used to improve secretion efficiency, the amino acid sequence of which is shown in SEQ ID NO.1.

The nucleotide sequence of the gene encoding the signal peptide that can be used to improve secretion efficiency is selected from any of the following sequences:

(a) the nucleotide sequence shown in SEQ ID NO.2 or its complementary sequence;

(b) obtained by performing one or more nucleotide substitutions, deletions or additions to the nucleotide sequence shown in SEQ ID NO. 2, and having the same function as the nucleotide sequence shown in SEQ ID NO. 2 The nucleotide sequence of the signal peptide function or its complement.

Recombinant expression vectors, recombinant genetically engineered bacteria, transgenic cell lines or expression cassettes containing any of the above-mentioned genes for signal peptides that can be used to improve secretion efficiency also belong to the protection scope of the present invention.

The recombinant genetically engineered bacteria is to fuse any of the above-mentioned genes of signal peptides that can be used to improve secretion efficiency to the N-terminus of the gene to be expressed, so that the N-terminus of the protein encoded by the fused gene is fused with a corresponding signal peptide; It is preferably a genetically engineered bacterium with improved ability to secrete β-galactosidase, or a genetically engineered bacterium capable of secreting transglutaminase; more preferably, a genetically engineered bacterium of Bacillus subtilis.

The application of the signal peptide that can be used for improving secretion efficiency in improving protein secretion and expression.

Said protein is thermostable beta-galactosidase or transglutaminase.

The secretory expression of the protein is secreted expression in a prokaryotic expression system; preferably secreted expression in Bacillus subtilis.

The Bacillus subtilis is preferably Bacillus subtilis ATCC6051 (B. subtilis ATCC6051).

Compared with the prior art, the present invention has the following advantages and effects:

1. The present invention constructs expression vectors that combine various signal peptides with the β-galactosidase encoding gene from the Bacillus subtilis homologous protein signal peptide library, and combines high-throughput screening methods to screen out the signal peptides that can improve β-galactosidase. - Signal peptide secreted by galactosidase. At the same time, an expression vector combining the signal peptide with the gene encoding the transglutaminase proenzyme is constructed to realize the secreted expression of the transglutaminase proenzyme.

2. The present invention provides an amino acid fragment, a signal peptide with strong secretion and expression activity, which can realize high expression of exogenous genes, and especially provides effective elements for Bacillus subtilis to express exogenous genes.

3. The present invention can increase the secretion of β-galactosidase by fusing the signal peptide, and further improve the enzymatic activity.

4. The present invention realizes the secretion and expression of transglutaminase proenzyme by fusing the signal peptide.

Description of drawings

Figure 1 is an electrophoresis diagram of the PCR product amplified by the biobrick in Example 1; wherein, lane M is the marker DNA, and lane 1 is the PCR amplification product of the biobrick.

FIG. 2 is a schematic diagram of the construction of plasmid pBEp43s-biobrick-bgaB and plasmid pBEp43-SP-bgaB in Example 1. FIG.

Figure 3 is the electrophoresis image of the amplified SPyocA fragment in Example 2; wherein, lane M: marker DNA; lane 1 is the SPyocA fragment.

Figure 4 is a schematic diagram of the construction of the expression plasmid pBEp43-SPyocA-proMTG in Example 2.

Figure 5 is the SDS-PAGE electrophoresis gel image of B.subtilis ATCC6051 (pBEp43-SPyocA-proMTG) expression in Example 2; wherein, lane M: marker; lane 1 is B.subtilis ATCC6051; lane 2 is B.subtilisATCC6051 (pBEp43 -SPyocA-proMTG); arrows indicate the position of target protein MTG.

Detailed ways

The present invention will be described in further detail below with reference to the examples, but the embodiments of the present invention are not limited thereto.

The molecular biology experimental techniques used in the following examples include PCR amplification, plasmid extraction, DNA fragment digestion, ligation, gel electrophoresis, etc. For details, please refer to "Molecular Cloning Experiment Guide" (Third Edition) (Sambrook J, Russell DW , Janssen K, Argentine J. Translated by Huang Peitang, etc., 2002, Beijing: Science Press).

Example 1 Screening of signal peptides secreted and expressed by β-galactosidase with high efficiency

Referring to the method of (Christian Degering et. Al, 2010), by constructing an expression vector combining various signal peptides with the β-galactosidase encoding gene (bgaB) from the Bacillus subtilis homologous protein signal peptide library, combined with A high-throughput screening method to obtain signal peptides that improve the secretion effect of β-galactosidase to varying degrees from clones, specifically including the following steps:

(1) Construction of pBEp43-biobrick-bgaB vector: the PCR fragment of biobrick (biological building block) obtained by annealing and extending two artificially synthesized fragments SEQ ID NO.3 and SEQ ID NO.4 has enzyme cleavage sites KpnI and XhoI, using The PCR product with a size of 100bp digested and purified by restriction endonuclease (as shown in Figure 1) was inserted into the plasmid pBE-P43-bgaB (according to the patent document: Pan Li et al. A DNA fragment with promoter function and application. CN201510074949.0 [P].2015. Construction) with the same endonuclease position to obtain pBEp43s-biobrick-bgaB plasmid (as shown in Figure 2), and the designed PCR fragment of biobrick is shown in SEQ ID NO.5. The base sequence of the amplified PCR fragment was confirmed by Sanger sequencing.

(2) Construction of pBEp43-SP (signal peptide database)-bgaB vector: extract the genomic DNA of Bacillus subtilis (Bacillus subttlis168, purchased from the Guangdong Provincial Microbial Culture Collection Center) (Omega Bacterial Genomic DNA Extraction Kit), and use the corresponding primers (F-SpyocA: 5′-GAGAGGAATGTCGACATGAAGAAAAAGAGAAAAGGCTGTT-3′; R-SpyocA: 5′-CGTTGTCCATCTCGAGGGCGATGACAAAGACAAAAATCAT-3′) to amplify the genomic DNA, and then the PCR product was recovered (Omega DNA gel recovery kit), after cloning transformation Insert into the above constructed pBEp43-biobrick-bgaB vector, use the restriction enzyme sites as KpnI and XhoI to construct the pBEp43-SP-bgaB vector (as shown in Figure 2).

(3) The constructed pBEp43-SP-bgaB vector was electrotransformed into Bacillus subtilis B.subtilisATCC6051, and the specific method was referred to non-patent literature records (Natalia P, Zakataeva, Oksana V et al. Asimple method to introduce marker-free genetic Modification into chromosome of naturally nontransformable Bacillus amyloliquefaciens strains [J]. Appl Microbiol Biotechnol. 2010, 85: 1201-1209), clones were obtained on resistant plates (kanamycin 20 μg/mL).

(4) The clones were picked and cultured in a 96-well plate, and the clones optimized for secretion were determined by measuring the enzymatic activity of β-glucogalactosidase. Determination of β-glucogalactosidase enzyme activity: Mix 32 μL of culture medium with 288 μL of 0.25% ONPG (o-Nitrophenyl-β-D-Galactopyranoside, o-nitrophenyl β-D-galactopyranoside) at 55°C After incubation for 15 min, the reaction was terminated by adding 320 μL of (w/w) Na ₂ CO ₃ . The reaction showed a color reaction, and the absorbance was measured at a wavelength of 405 nm.

(5) Sequence the secretion-optimized clones to determine whether the signal peptide has been modified. The signal peptide that can improve the secretion effect of β-galactosidase is obtained through the above-mentioned steps. After the NCBI check, it is the signal peptide of the yocA gene. The amino acid sequence is: MKKKRKGCFAAAGFMMIFVFVIA (SEQ ID NO. 1), and the nucleoside encoding the amino acid sequence The acid sequence is: ATGAAGAAAAAGAGAAAAGGCT GTTTCGCTGCTGCGGGTTTTATGATGATTTTTGTCTTTGTCATCGCC (SEQ ID NO. 2).

Embodiment 2 Detection of signal peptide expression level

(1) Construction of MTG (transglutaminase) extracellular expression plasmid: using plasmid pBEp43-proMTG (according to patent document: Pan Li et al. A recombinant Bacillus subtilis and its method for producing transglutaminase. CN201210052578 .2[P].2012. Construction) is an expression plasmid, using the pBEp43-SPyocA-bgaB plasmid obtained in the above Example 1 as a template, primers F-SPyocA (5′-GAGAGGAATGTCGACATGAAGAAAAAGAGAAAAGGCTGTT-3′), R-SPyocA (5′) -CGTTGTCCATCTCGAGGGCGATGACAAAGACAAAAATCAT-3') to amplify an approximately 100 bp SPyocA fragment (see Figure 3). The signal peptide (SPyocA fragment) and the plasmid (pBEp43-proMTG) were connected by the In-fusion method (for the specific operation method, see the HiFi DNA Assembly Master Mix of NEBuilder Company) to construct the extracellular plasmid pBEp43-SPyocA-proMTG for MTG expression (see Fig. 4). First, it was transformed into Escherichia coli (E.coli JM110) by chemical transformation method to obtain positive clones. After sequencing, the plasmids were extracted and transformed into Bacillus subtilis B.subtilis ATCC6051 by electrotransformation method.

(2) The obtained transformants B.subtilis ATCC6051 (pBEp43-SPyocA-bgaB) and B.subtilis ATCC6051 (pBEp43-SPyocA-proMTG) were cultured in 10 mL of LB medium (kanamycin 20 μg/mL) at 37° C., Activated at 200rpm for 12h, inoculated the activated seed liquid in 50mL LB medium (kanamycin 20μg/mL, 1% (w/w) glucose) inoculation amount of 1% (volume ratio), 37°C, 200rpm total fermentation 48h, sampling every 6 hours.

(3) Determination of the enzyme activity of β-glucosidase: The results showed that the expression of β-glucosidase secreted by the signal peptide SPyocA, the expression of the enzyme activity was the highest at 30-48h, of which the highest enzyme activity was 5.76U/hour at 36h. mL.

(4) SDS-PAGE: Centrifuge the fermentation broth of B.subtilis ATCC6051 (pBEp43-SPyocA-proMTG) for 36h to take the supernatant, run SDS-PAGE electrophoresis, and compare it with wild-type B.subtilis ATCC6051 as B.subtilis ATCC6051. For the control, the protein gel map showed that the band at 44-46KDa was consistent with the size of MTG protein, while the wild-type control had no band in this size range. The experimental results indicated that MTG protease was expressed (see Figure 5). It indicated that SPyocA could act as an effective signal peptide on Bacillus subtilis.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

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Claims (3)

1. the application of a signal peptide that can be used to improve secretion efficiency in improving protein secretion expression, it is characterized in that: the amino acid sequence of described signal peptide is as shown in SEQ ID NO.1; Described protein is thermostable beta-galactosidase or transglutaminase; Said protein is secreted and expressed in Bacillus subtilis. 2. The application according to claim 1, wherein the nucleotide sequence of the signal peptide that can be used to improve secretion efficiency is selected from the following sequences: the nucleotide sequence shown in SEQ ID NO.2 or its complementary sequence. 3. application according to claim 2 is characterized in that: the described gene of the signal peptide that can be used for improving secretion efficiency is fused to the N-terminus of the gene to be expressed, and the N-terminus of the protein encoded by the fused gene is fused. There are corresponding signal peptides.

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