Non-classical secretory protein and application thereof to protein secretory expression

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

Active Publication Date: 2016-05-25
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Of course, the detection of non-canonical secreted proteins in the extracellular e...

Method used

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  • Non-classical secretory protein and application thereof to protein secretory expression
  • Non-classical secretory protein and application thereof to protein secretory expression
  • Non-classical secretory protein and application thereof to protein secretory expression

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Experimental program
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Embodiment 1

[0034] The secretory expression of embodiment 1RDPE in Bacillus subtilis

[0035] According to the sequence of the DPEase coding gene rdpe derived from Ruminococcussp. Subsequent expression vector construction. The synthesized gene was connected to the pUC57 vector and named pUC57-RDPE.

[0036] Plasmids pUC57-RDPE and pMA5 were subjected to NdeI and BamHI double enzyme digestion treatment respectively; the double enzyme digestion reaction conditions were 37°C water bath for 3h. The double-digested products were recovered by gel respectively, and the rdpe fragment with NdeI and BamHI restriction sites at both ends and the linearized pMA5 vector were obtained, and the two were ligated with T4 ligase at room temperature for 30 minutes; the ligated product was transformed Colony competent cells DH5α were screened for resistance with ampicillin (100 μg / mL), positive clones were screened by colony PCR, and plasmids were extracted for sequencing verification. For plasmid constr...

Embodiment 2

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

[0040] Using the signal peptide prediction software SignalP4.1 (http: / / www.cbs.dtu.dk / services / SignalP / ) to analyze the amino acid sequence of RDPE, it was found that there was no typical signal peptide at the N-terminus of RDPE, and there were no typical signal peptides in other regions. No secretion signal sequence is present.

Embodiment 3

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

[0042] The construction method of this partial recombinant plasmid is based on the method in the literature (You et al. ApplEnvironMicrobiol2012, 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 (ProlongedoverlapextensionPCR), 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 constructed pl...

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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 is an important protein-producing strain in industrial production. Bacillus subtilis has attracted the attention of many scientific researchers because of its strong protein secretion ability, GRAS (generally recognized as safe), genetic ease of operation, no codon preference, and low fermentation cost. 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 proteins are transported through the ABC transporter ...

Claims

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Application Information

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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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