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Amphiphilic alpha helix self-assembling peptide and application thereof

A technology selected from, intein, applied in the field of genetic engineering, can solve the problems of restricting the application of inclusion body expression form, complex technology, low yield, etc.

Active Publication Date: 2014-12-31
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the protein refolding technology still has problems such as high cost, low yield, and technical complexity [3], which greatly limits the application of inclusion body expression in actual production

Method used

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  • Amphiphilic alpha helix self-assembling peptide and application thereof
  • Amphiphilic alpha helix self-assembling peptide and application thereof
  • Amphiphilic alpha helix self-assembling peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Construction of a fusion protein expression vector using Bacillus subtilis lipase A (LipA) and green fluorescent protein (GFP) as target proteins

[0066] 1.1 Amplification of polynucleotide fragments of 18A variants (19 species)

[0067] Firstly, the nucleotide sequence of the PT-type linker and the 18A variant was designed with the online tool DNAworks. The oligonucleotide primers shown in Table 2 were designed by DNAWorks [13] and synthesized, and then the complete sequence encoding the 18A variant (Hind III-linker-18A variant-Xho I) was obtained by overlapping PCR (overlapping PCR) method. polynucleotide sequence.

[0068] Table 2 is used to amplify the primer list of 18A variant

[0069]

[0070]

[0071]

[0072] a The underlined parts of the primers are the recognition sites of restriction endonucleases Hind III and Xho I, respectively.

[0073] Taking one of the variants, 18Av1, as an example, the specific method for amplifying the polynu...

Embodiment 2

[0093] Example 2: Expression, cell growth status and enzyme activity assay of a fusion protein with Bacillus subtilis lipase A (LipA) as the target protein

[0094] 2.1 Induced expression of fusion protein

[0095] The strain constructed in Example 1 (containing plasmids pET-30a(+)-LipA-native, pET-30a(+)-GFP-18A and pET-30a(+)-GFP-18A variants) was inoculated into the 50 μg / mL kanamycin in LB liquid medium, and cultured in a shaker at 37°C to logarithmic phase (OD 600 =0.4-0.6), add 0.2mM IPTG, induce at 30℃ for 6 hours, harvest the cells, and measure the bacterial concentration OD 600 (The following will be 1mL of OD 600 The amount of cells equal to 1 is called 1OD).

[0096] 2.2 Cell growth status

[0097] The growth state of the cells expressing the LipA-18A variant fusion protein is shown in Table 7 and Figure 4 As shown in A.

[0098] Table 7 expresses the OD of LipA-18A and LipA-18A variant fusion protein cells 600 value

[0099] 18A variant LipA-18...

Embodiment 3

[0107] Example 3: Expression of fusion protein with green fluorescent protein (GFP) as the target protein and its intracellular distribution

[0108] The strain constructed in Example 1 was inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultivated in a shaker at 37°C to logarithmic phase (OD 600 =0.4-0.6), add 0.2mM IPTG, induce at 23°C for 22 hours, and harvest the cells.

[0109] The harvested cells were treated with 4% paraformaldehyde at 4°C for 1 h. Fluorescent confocal microscopy of GFP cells was performed on a Zeiss710 inverted confocal microscope (Zeiss LSM710confocal microscope) with an excitation wavelength of 488nm.

[0110] The intracellular distribution of active enzyme aggregates induced by 18A and 18A variants was as follows Figure 5 shown. It can be clearly seen from the fluorescent photos that the fluorescence of cells expressing GFP-18A fusion protein is mainly distributed on the inner side of the cell membrane. It shows that the GFP...

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Abstract

The invention relates to an amphiphilic alpha helix self-assembling peptide derived from SEQ ID NO:1 and an application of the amphiphilic alpha helix self-assembling peptide in protein production and purification. When the amphiphilic alpha helix self-assembling peptide and target protein are subjected to fusion expression, the amphiphilic alpha helix self-assembling peptide can induce the fusion protein to form active aggregate in cell, by comparing with polypeptide in SEQ ID NO: 1, the spatial distribution of the formed active aggregate in the cell is changed, a host cell growth state is improved, and fusion protein output is increased.

Description

technical field [0001] The invention relates to the field of genetic engineering. In particular, the present invention relates to novel amphipathic alpha-helical self-assembling peptides and their use in protein production and purification. Background technique [0002] With the increasing demand for protein and polypeptide products such as enzyme preparations and protein drugs, the use of prokaryotic systems to express recombinant proteins has become more and more widely used in industrial biotechnology. When expressing foreign genes in prokaryotic systems, the target protein tends to exist in the form of insoluble protein aggregates (also called inclusion bodies)[1]. Traditionally, inclusion bodies refer to insoluble precipitates formed by misfolding polypeptides that have no biological activity [2]. However, protein expression in the form of inclusion bodies has many advantages, such as large protein expression, high purity, easy separation and operation, avoiding prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K19/00C12N15/11C12N15/62C12N15/63C12N1/21C12P21/00
Inventor 林章凛周碧红邢磊赵青
Owner TSINGHUA UNIV
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