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

Abstract

The invention discloses a signal peptide capable of improving secretion efficiency and an application of the peptide. The amino acid sequence of the signal peptide is as shown in SEQ ID NO.1. The nucleotide sequence for encoding the signal peptide is selected from any one of the following sequences: (a) a nucleotide sequence shown in SEQ ID NO.2 or a complementary sequence of the nucleotide sequence, and (b) a nucleotide sequence which is obtained by substitution, missing or adding of one or more nucleotides on the nucleotide sequence shown in the SEQ ID NO.2, has a same nucleotide sequence with the nucleotide sequence as shown in the SEQ ID NO.2, and used as a signal functional nucleotide sequence, or a complementary sequence of the obtained nucleotide sequence. The invention provides thesignal peptide with strong secretory expression activity, the high expression of exogenous genes can be realized, and an effective element is provided for exogenous gene expression for the bacillus subtilis.

Description

technical field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a signal peptide which can be used to improve secretion efficiency and its application. Background technique

[0002] Bacillus subtilis is a Gram-positive aerobic bacillus widely used in industrial enzyme production. Compared with other prokaryotes, using Bacillus subtilis as a host for protein expression has the following advantages: 1. Clear genetic background and complete genome sequencing; 2. No codon preference; 3. Simple fermentation and no special requirements for nutrition ; 4. Possess a high-efficiency protein secretion system, and most of the proteins secreted by it have biological activity and natural conformation; 5. No pathogenicity and no endotoxin.

[0003] In Bacillus subtilis, there have been more in-depth studies on the expression secretion system and related secretion elements, and the screening of signal peptides is an effective strate...

Examples

Embodiment 1

[0029] Example 1 Screening of Signal Peptides Efficiently Secreted and Expressed by β-galactosidase

[0030] With reference to the method of (Christian Degering et.Al, 2010), by constructing the expression vector that various signal peptides combine with β-galactosidase coding gene (bgaB) from Bacillus subtilis homologous protein signal peptide library, combine 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:

[0031] (1) Construction of pBEp43-biobrick-bgaB vector: The PCR fragment of biobrick (biological building block) obtained by annealing and extending two artificially synthesized fragments of SEQ ID NO.3 and SEQ ID NO.4 has restriction sites KpnI and XhoI, and used Restriction endonuclease digested and purified 100bp PCR product (such as figure 1 ) into the same endonuclease position of the plasmid pBE-P43-bgaB (according to the pa...

Embodiment 2

[0036] The detection of embodiment 2 signal peptide expression levels

[0037] (1) Construction of MTG (transglutaminase) extracellular expression plasmid: the plasmid pBEp43-proMTG (according to the patent literature: Pan Li et al. A recombinant Bacillus subtilis and its method for producing transglutaminase. CN201210052578 .2[P].2012.Construction) as an expression plasmid, using the pBEp43-SPyocA-bgaB plasmid obtained in Example 1 above as a template, primers F-SPyocA (5'-GAGAGGAATGTCGACATGAAGAAAAAGAGAAAAGGCTGTT-3'), R-SPyocA (5' -CGTTGTCCATCTCGAGGGCGATGACAAAGACAAAAATCAT-3′) amplifies about 100bp SPyocA fragment (see image 3 ). The signal peptide (SPyocA fragment) and the plasmid (pBEp43-proMTG) were connected by the In-fusion method (see the HiFi DNA Assembly Master Mix of NEBuilder for the specific operation method), and the MTG expression extracellular plasmid pBEp43-SPyocA-proMTG was constructed (see Figure 4 ). First transform into Escherichia coli (E.coli JM110) b...

technical field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a signal peptide which can be used to improve secretion efficiency and its application. Background technique

[0002] Bacillus subtilis is a Gram-positive aerobic bacillus widely used in industrial enzyme production. Compared with other prokaryotes, using Bacillus subtilis as a host for protein expression has the following advantages: 1. Clear genetic background and complete genome sequencing; 2. No codon preference; 3. Simple fermentation and no special requirements for nutrition ; 4. Possess a high-efficiency protein secretion system, and most of the proteins secreted by it have biological activity and natural conformation; 5. No pathogenicity and no endotoxin.

[0003] In Bacillus subtilis, there have been more in-depth studies on the expression secretion system and related secretion elements, and the screening of signal peptides is an effective strate...

Examples

Embodiment 1

[0029] Example 1 Screening of Signal Peptides Efficiently Secreted and Expressed by β-galactosidase

[0030] With reference to the method of (Christian Degering et.Al, 2010), by constructing the expression vector that various signal peptides combine with β-galactosidase coding gene (bgaB) from Bacillus subtilis homologous protein signal peptide library, combine 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:

[0031] (1) Construction of pBEp43-biobrick-bgaB vector: The PCR fragment of biobrick (biological building block) obtained by annealing and extending two artificially synthesized fragments of SEQ ID NO.3 and SEQ ID NO.4 has restriction sites KpnI and XhoI, and used Restriction endonuclease digested and purified 100bp PCR product (such as figure 1 ) into the same endonuclease position of the plasmid pBE-P43-bgaB (according to the pa...

Embodiment 2

[0036] The detection of embodiment 2 signal peptide expression levels

[0037] (1) Construction of MTG (transglutaminase) extracellular expression plasmid: the plasmid pBEp43-proMTG (according to the patent literature: Pan Li et al. A recombinant Bacillus subtilis and its method for producing transglutaminase. CN201210052578 .2[P].2012.Construction) as an expression plasmid, using the pBEp43-SPyocA-bgaB plasmid obtained in Example 1 above as a template, primers F-SPyocA (5'-GAGAGGAATGTCGACATGAAGAAAAAGAGAAAAGGCTGTT-3'), R-SPyocA (5' -CGTTGTCCATCTCGAGGGCGATGACAAAGACAAAAATCAT-3′) amplifies about 100bp SPyocA fragment (see image 3 ). The signal peptide (SPyocA fragment) and the plasmid (pBEp43-proMTG) were connected by the In-fusion method (see the HiFi DNA Assembly Master Mix of NEBuilder for the specific operation method), and the MTG expression extracellular plasmid pBEp43-SPyocA-proMTG was constructed (see Figure 4 ). First transform into Escherichia coli (E.coli JM110) b...

Images