A signal peptide that can effectively improve the efficiency of protein secretion and expression and its application

A technology for secretion expression and signal peptide, applied in the field of signal peptide, can solve the problem of not being able to achieve better technical effects, and achieve the effect of improving secretion efficiency

Active Publication Date: 2019-04-30
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the secretory expression of Escherichia coli has received extensive attention, and there are many types of signal peptides, these signal peptide sequences have not achieved good technical results when used to express phospholipase D in Escherichia coli

Method used

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  • A signal peptide that can effectively improve the efficiency of protein secretion and expression and its application
  • A signal peptide that can effectively improve the efficiency of protein secretion and expression and its application
  • A signal peptide that can effectively improve the efficiency of protein secretion and expression and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: A new type of signal peptide

[0043] The nucleotide sequence of the signal peptide provided in the present invention is as shown in SEQ ID NO: 2, which is based on the signal peptide PelB (SEQ ID NO: 4) and OmpA (SEQ ID NO: 5). It was transformed. The N terminal of the signal peptide uses the first three amino acid disabled MKK of the signal peptide OmpA, the middle sequence is the strong hydrophobic sequence LLPTAAAGLLLLAAQP of PelB, and the C terminal uses the last three amino acid residues AQA of OmpA. The signal peptide sequence is obtained by chemical synthesis or PCR, and its usability is analyzed by signalP 3.0.

[0044] This signal peptide was cloned into the NcoI restriction site of the vector pET-28a(+), and then the target gene PLD was cloned into the C-terminal of this signal peptide, the secretion of the target protein was effectively enhanced after expression. The secretion of PLD is effectively increased to 0.23g / L, and the extracellular enzyme a...

Embodiment 2

[0045] Example 2: Acquisition of phospholipase D gene.

[0046] The target protein used in the present invention is phospholipase D in Escherichia coli BL21, and its phospholipase D functional gene is obtained by PCR.

[0047] 1. Genomic extraction of E. coli BL21.

[0048] 1) Collect 1ml of bacterial solution in 1.5ml Tube, centrifuge at 12,000rpm for 2 minutes, and discard the supernatant (cell culture solution).

[0049] 2) Add 180μl of Buffer GL, 20μl of Proteinase K (20mg / ml) and 10μl of RNase A (10mg / ml) to mix thoroughly and incubate in a water bath at 56℃ for 10 minutes. At this time, the solution should be transparent and clear. .

[0050] 3) Add 200 μl of Buffer GB and 200 μl of 100% ethanol, and mix thoroughly by pipetting.

[0051] 4) Install the Spin Column on the Collection Tube, transfer the solution to the Spin Column, centrifuge at 12,000 rpm for 2 minutes, and discard the filtrate.

[0052] 5) Add 500 μl of Buffer WA to Spin Column, centrifuge at 12,000 rpm for 1 minute...

Embodiment 3

[0064] Example 3: Construction of a phospholipase D secretory expression strain to induce expression.

[0065] 1. Construction and transformation of recombinant plasmids.

[0066] First, the plasmid pET-28a was linearized with EcoRI and NdeI, and purified and recovered. The PCR product and linearized plasmid are ligated by one-step cloning method. The system is as follows: PCR purified product 2ul, linearized pET-28a plasmid 4ul, CE buffer 4ul, ExnaseⅡ 2ul, deionized water 8ul, react at 37℃ for 30 minutes , Construct the recombinant plasmid pET-28a-PLD. Then the above plasmid pET-28a-PLD was treated with NcoI endonuclease. Finally, the synthesized signal peptide sequence and linearized pET-28a-PLD are ligated by one-step cloning method to construct the final plasmid pET-28a-sp-PLD ( Figure 4 ). Send Jin Weizhi sequencing.

[0067] 2. Induced expression of recombinant bacteria.

[0068] The secretion expression plasmid pET-28a-sp-PLD constructed above was transferred into the Rose...

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Abstract

The invention discloses a signal peptide for mediating protein secretion expression and belongs to the field of protein engineering and gene engineering. By modifying known signal peptides PelB and OmpA, the N terminal of the signal peptide contains the first three amino acid residues MKK of the signal peptide OmpA, the C terminal of the signal peptide contains the last three amino acid residues AQA of the signal peptide OmpA, and a middle hydrophobic structure is the hydrophobic sequence LLPTAAAGLLLLAAQP of the signal peptide PelB. After the signal peptide capable of effectively improving protein secretion efficiency is used, the secretion expression capability of a strain on target protein is obviously improved; the extracellular enzyme activity of phospholipase D of target protein can be improved by 2 times, the extracellular enzyme production capability of the modified strain is significantly improved, and industrialized application is better facilitated.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a signal peptide that can effectively improve the efficiency of protein secretion and expression and its application. Background technique [0002] Phospholipase D (EC3.1.4.4) is a special ester hydrolase, which can catalyze the hydrolysis of phosphoric acid and organic alkali (such as choline, ethanolamine, etc.) in the phospholipid molecule to form an ester bond, which is 4 times the phospholipid key. In addition to hydrolysis, it can also catalyze the binding of various hydroxyl-containing compounds to the bases of phospholipids under specific conditions to form new phospholipids. This property is called the phospholipid transfer property of phospholipase D, and is also commonly referred to as alkali. Radical exchange reaction. Using this feature, phospholipids can be modified to prepare high-purity single phospholipids and rare phospholipids, which has ver...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12N15/11C12N1/21C12N15/70C12N15/67C12N9/16C12R1/19
CPCC07K14/00C12N9/16C12N15/67C12N15/70C12N2800/101C12Y301/04004
Inventor 应汉杰徐煜陈勇朱杰柳东牛欢清吴菁岚朱晨杰庄伟
Owner NANJING TECH UNIV
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