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Bacillus subtilis self-induced expression system and application thereof

A Bacillus subtilis, gene expression technology, applied in the field of promoter engineering, can solve the problems of unsatisfactory industrial production and low heterologous protein expression

Inactive Publication Date: 2017-08-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the self-inducible expression system has a good application prospect because it does not need to add inducers and can save the cost of industrial production, but the self-inducible promoter expresses the problem of low expression of heterologous proteins, so it cannot meet the needs of industrial production

Method used

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  • Bacillus subtilis self-induced expression system and application thereof
  • Bacillus subtilis self-induced expression system and application thereof
  • Bacillus subtilis self-induced expression system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Construction of the shuttle vector pBSG01 containing the promoter srfA and the target protein green fluorescent protein GFP.

[0021] (1) Construction of the shuttle vector pBSG: using the plasmid pUC19 as a template, using P1 / P2 as primers, amplifying the 2.2kb fragment 1 containing the resistance gene bla and the replication origin site sequence pBR322ori; using the plasmid pMA09 as a template, Using P3 / P4 as primers, amplify fragment 2 containing replication-related protein Rep, resistance gene neor and ble; connect fragments 1 and 2 by SLIC method, transform E.coli, pick positive clones for sequencing, and obtain E. coli-B.subtilis shuttle cloning vector pBSG.

[0022] (2) Construction of the expression vector pBSG0: using the B. subtilis 168 genome as a template, primers P5 / P6 were used to amplify the promoter PsrfA fragment; primers P7 / P8 were used to obtain linear pBSG; PsrfA and linear pBSG were connected by SLIC method to obtain Vector pBSG0.

[002...

Embodiment 2

[0026] Example 2 Deletion of upstream elements to different degrees to obtain 4 promoters with different strengths

[0027] Four pairs of different primers (F1-up, P srfA -down; P2-up, P srfA -down; P3-up, P srfA -down; P4-up, P srfA-down), the promoter P01 (i.e. PsrfA) in the original vector pBSG01 was truncated to different lengths, and four truncated promoters P02, P03, P04, P05 (SEQ ID NO.34~37) were obtained, which were respectively Transformed into Bacillus subtilis 168, and then cultured in shake flasks (see "Culture Conditions" above for culture conditions), see the truncation schematic diagram and expression status in figure 1 . Among them, P02, P03, and P04 expressed GFP yields higher than P01, and P04 increased the most obviously, increasing by 20%; while P05 expressed GFP yields lower than the original P01, and its yield dropped to 50% of the original.

Embodiment 3

[0028] Rational design of embodiment 3 promoter

[0029] The core region of the promoter P04 with obvious truncation effect in Example 2 was rationally designed, and 7 pairs of different primers were designed (P1-up, P1-down; P2-up, P2-down; P3-up, P3-down; P4-up, P4-down; P5-up, P5-down; P6-up, P6-down; P7-up, P7-down), mainly the core region of the promoter -35, -15 and The -10 region was mutated into a conserved region, and seven promoters (P10, P11, P12, P13, P14, P15, P16) (SEQ ID NO.38-44) were constructed. figure 2 . The recombinant Bacillus subtilis constructed with these seven promoters expressed the green fluorescent protein GFP. The GFP expression intensity of the obtained seven recombinant strains was higher than that of the original strain (BS168-pBSG01-GFP), among which P11 (-35 region changed to the conservative region TTGACA) had the most obvious effect, and its expression level increased by 150% compared with the original promoter.

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Abstract

The invention discloses a bacillus subtilis self-induced expression system and an application thereof, and belongs to the field of promoter engineering. On the basis of rational design on a promoter srfA and by mutating core regions, namely No.35 region and No.10 region, of the promoter into a conserved region, some relatively strong promoters are obtained; and with green fluorescent protein GFP as a reporter gene, the mutated promoters are 150% higher than the original promoter in highest expression amount. Subsequently, aminopeptidase AP expression and nattokinase NK expression are conducted, so that mutant expression amounts, in comparison with that of the original promoter, are improved by 360% and 50%. The method provided by the invention, in comparison with random mutation, greatly reduces workload and is effective; and meanwhile, as the strategy is applied to transformation of other promoters, the capacity of the transformed promoters in expressing a target gene is also greatly improved.

Description

technical field [0001] The invention relates to a Bacillus subtilis self-induced expression system and application thereof, belonging to the field of promoter engineering. Background technique [0002] Amionpeptidases (AP) are a class of exoproteases that can selectively remove amino acid residues from the N-terminus of proteins and polypeptide chains, and have broad market prospects in the food, pharmaceutical, and chemical industries. At present, the commercialized aminopeptidase is mainly produced by wild bacteria. Since the enzyme-producing ability of wild bacteria is low, the price of aminopeptidase is relatively expensive, so it is of great significance to increase the production of aminopeptidase. [0003] Nattokinase and its expression status: Nattokinase (Nattokinase, NK) is an alkaline serine protease produced by Bacillus natto or Bacillus subtilis natto. Because of its special thrombolytic activity, it can prevent and treat blood vessels. Compared with other thro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/75C12N1/21C12R1/125
Inventor 周哲敏程锦涛崔文璟韩来闯缪胜男姚远
Owner JIANGNAN UNIV
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