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A non-canonical secretory protein and its application in protein secretory expression

A technology of secreting protein and secreting expression, which is applied in the field of genetic engineering and can solve problems such as cytoplasmic protein leakage

Active Publication Date: 2019-01-08
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Of course, the detection of non-canonical secreted proteins in the extracellular environment can easily be attributed to the leakage of cytoplasmic proteins by cell lysis

Method used

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  • A non-canonical secretory protein and its application in protein secretory expression
  • A non-canonical secretory protein and its application in protein secretory expression
  • A non-canonical secretory protein and its application in protein secretory expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Secretion and expression of RDPE in Bacillus subtilis

[0035] According to the source of rumen bacteria ( Ruminococcus sp. 5_1_39B_FAA) DPEase encoding gene rdpe The whole gene synthesis is carried out, and the 5' and 3' ends of the nucleotide sequence of the gene are respectively introduced Nde I and Bam HI restriction enzyme cutting site to facilitate the subsequent construction of expression vectors. The synthesized gene was connected to the pUC57 vector and named pUC57-RDPE.

[0036] Plasmids pUC57-RDPE and pMA5 were carried out separately Nde I and Bam HI double enzyme digestion treatment; the reaction condition of double enzyme digestion is 37°C water bath for 3 h. The double-digested products were recovered by gel respectively to obtain Nde I and Bam HI restriction site rdpe The fragment and the linearized pMA5 vector were ligated with T4 ligase for 30 min at room temperature; the ligation product was transformed into colony competen...

Embodiment 2

[0039] Example 2 SignalP predicts the signal peptide or signal sequence of RDPE

[0040] Using the signal peptide prediction software SignalP 4.1 (http: / / www.cbs.dtu.dk / services / SignalP / ) to analyze the amino acid sequence of RDPE, it was found that there is no typical signal peptide at the N-terminal of RDPE, nor in other regions. Any secretion signal sequence is present.

Embodiment 3

[0041] Example 3 RDPE cannot be secreted through the Sec and Tat secretion pathways

[0042] The partial recombinant plasmid construction method is based on the method in the literature (You et al. Appl EnvironMicrobiol 2012, 78:1593-5). The four Sec signal peptides (SP sacB 、SP AprE 、SP AmyL and SP AmyE , The nucleic acid sequences are respectively shown in SEQ ID NO. 2, 3, 4 and 5), and the PCR products obtained by cloning were respectively gel-recovered. Using pMA5-RDPE as a template, primers were used to amplify the linearized vector pMA5-RDPE, and the resulting PCR product was gel-recovered. The above four signal peptide fragments and the linearized vector pMA5-RDPE were subjected to POE-PCR (Prolonged overlap extension PCR), and the obtained fusion products were directly transformed into colon competent cells DH5α, respectively, to obtain recombinant expression plasmids pMA5-SP sacB -RDPE, pMA5-SP AprE -RDPE, pMA5-SP AmyL -RDPE and pMA5-SP AmyE -RDPE. The constr...

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Abstract

The invention relates to heterologous protein secreted in bacillus subtilis in a non-classical path and application thereof to protein secretory expression. Carrying no signal peptide or secretory signals, D-allulose3-epimerase RDPE from Ruminococcus sp.5-139B-FAA can be secreted out of cells in quantity; it is proved through experiments that RDPE is not secreted in an Sec or Tat path or leaked out of the cells due to cell autolysis and lysis. Therefore RDPE is non-classical secretory protein in the bacillus subtilis. RDPE is respectively fused with 18 kinds of target protein, an attempt is made to use RDPE as export signals for achieving secretory expression of the target protein, 16 kinds of fused protein are obviously expressed in the cells, 9 kinds of fused protein are successfully secreted out of the cells, and the secretion level of two kinds of fused protein accounts for more than 50% of the corresponding total protein. Therefore, a new strategy for achieving protein secretory expression in the bacillus subtilis is developed.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a heterologous protein secreted through a non-classical secretion pathway in Bacillus subtilis, and a novel strategy for using the protein as a transport signal to realize the secretion and expression of the protein in Bacillus subtilis. Background technique [0002] Bacillus subtilis ( Bacillus subtilis ) is an important protein-producing strain in industrial production. Because of its strong protein secretion ability, location of safe strains (GRAS, generally recognized as safe), easy genetic manipulation, no codon preference, and low fermentation cost, Bacillus subtilis has attracted the attention of many scientific researchers. In Bacillus subtilis, most proteins are transported out of the cell through the Sec pathway in an unfolded state, a few proteins (such as PhoD and YwbN) are secreted into the medium through the Tat pathway in a folded state, and some pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/75C12N15/65
CPCC12N9/90C12N15/65C12N15/75C12N2800/101C12Y503/01
Inventor 张大伟陈景奇赵留群孙际宾
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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