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Elastin-like polypeptide elp for prokaryotic expression of fusion protein prx by non-enzymatic non-chromatographic purification method

A technology similar to elastin and fusion protein, applied in the field of genetic engineering, to achieve good application prospects, reduce impact, and improve elastic function

Active Publication Date: 2017-10-10
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, PrxI has become an important target for screening anti-tumor drugs. Therefore, prokaryotic expression of PrxI is of great significance for establishing a drug screening platform and screening anti-tumor drugs. At present, there is no non-enzymatic and non-chromatographic purification method for prokaryotic expression of Prx I related reports

Method used

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  • Elastin-like polypeptide elp for prokaryotic expression of fusion protein prx by non-enzymatic non-chromatographic purification method
  • Elastin-like polypeptide elp for prokaryotic expression of fusion protein prx by non-enzymatic non-chromatographic purification method
  • Elastin-like polypeptide elp for prokaryotic expression of fusion protein prx by non-enzymatic non-chromatographic purification method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] combined with figure 1 Describe the method of preparation.

[0039] 1. Construction of promoter protein, precursor protein expression vector and host cell

[0040] (a) Synthesis of ELP-IN, ELP-IC and PrxI genes

[0041] The primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and the DNAs of ELP-IN and ELP-IC (SEQ ID NO: 1 and SEQ ID NO: 3) were obtained by PCR. The primer sequences are as follows:

[0042] ELP-IN:

[0043] 5' primer:

[0044] 5'GG CCATGG CCGTGCCTGGTAAAGGAGTTCC3' (Nco Ⅰ)

[0045] 3' primer:

[0046] 5'GCGC AAGCTT ATTCTTTCACATACAGGCACATGCC3'(HindⅢ)

[0047] ELP-IC:

[0048] 5' primer:

[0049] 5'CC CATATG GTGCCTGGTAAAGGAG3' (NdeI)

[0050] 3' primer:

[0051] 5'TTAAGGATCCGCTGTTATGGGTCAGAATATCGTT3'(BamH Ⅰ)

[0052] In addition, two primers were designed and synthesized for PCR cloning the cDNA of Prx Ⅰ. The primer sequences are as follows:

[0053] 5' primer:

[0054] 5'CG GGATCC ATGTCTTCAGGAAATG...

Embodiment 2

[0072] The groping of the indirect condition of embodiment 2 object protein

[0073] (a) Incubate for 4 hours under different temperature conditions

[0074] After the purified protein was incubated at different temperatures (4°C, 10°C, 15°C, 20°C, 25°C) for different durations (10min, 20min, 30min, 1h, 2h, 4h) at a ratio of 1:1, SDS- PAGE electrophoresis detection, drawing the relationship curve of self-cutting percentage and time under different temperature conditions ( Figure 7 ). Protein self-cleavage is relatively complete at 25°C. In order to reduce the impact of high temperature on the activity of the target protein, 25°C is temporarily used to induce self-cleavage.

[0075]

[0076] 1×Reaction buffer: (50mM Tris, 100mM NaCl, 5% Glycerol)

[0077] (b) 25°C water bath induces protein splicing

[0078] The promoter protein and the precursor protein were mixed at a ratio of 1:1, and the self-cleavage was induced for different periods of time (1min, 5min, 10min, 0.5...

Embodiment 3

[0079] Purification and activity detection of embodiment 3 target protein

[0080] (a) Purification of target protein

[0081] According to the system of Example 2, the purified protein was induced to self-cleavage of the precursor protein for 0.5 h at an optimum temperature of 25° C., and then purified by ITC method to obtain the target protein PrxI ( Figure 9 ).

[0082] (b) Activity detection of PrxI

[0083] The PrxI activity prepared above was measured by referring to the PrxI activity detection method mentioned in the document Adenanthin targets peroxiredoxin I and II to induce differentiation of leukemia cells. Detect the A340 value at different time points with BioTek Synergy2 multifunctional microplate reader (Molecular Devices, USA), draw the curve of A340 value changing with time ( Figure 10 ).

[0084] Results: The activity of the sample was comparable to that of the standard, and the whole purification process did not affect the activity of the protein. Thi...

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Abstract

The invention discloses an elastin-like polypeptide ELP used for prokaryotic expression of fusion protein Prx in a non-enzyme-cut and non-chromatographic purification method, belonging to the field of genetic engineering. The invention changes the type of the fourth amino acid in the elastin-like pentapeptide unit to improve its elastic function, thereby making it possible to reduce the length of ELP. The ratio of K, V and F in the fourth base in the ELP pentapeptide unit is 1:6:3. In terms of intein, the present invention uses high-efficiency intein gp41-1, the C-terminus and N-terminus of the intein are expressed separately, and its internal gene is mutated. The elastin-like polypeptide ELP is ELP-IN and precursor protein ELP-IC-PrxI. Utilizing the elastic function of elastin for non-chromatographic purification of protein PrxI, without using chromatographic column separation, the target protein will precipitate after incubation at a certain temperature, and the precipitate can be re-dissolved in the buffer solution, which can make protein purification more efficient and faster.

Description

technical field [0001] The invention relates to an elastin-like polypeptide ELP, in particular to an elastin-like polypeptide ELP used for prokaryotic expression of a fusion protein Prx in a non-enzymatic and non-chromatographic purification method, and belongs to the field of genetic engineering. Background technique [0002] Non-enzymatic non-chromatographic purification technology is a method of separating and purifying fusion proteins by genetic engineering. Traditional protein purification relies on chromatographic purification. The equipment used in this method is expensive, which makes the protein production cost high; after purification, the target protein containing the fusion protein needs a tool enzyme (such as enterokinase) to remove the label. The enzyme has high production cost and poor specificity. . Intein-mediated self-cleavage makes it unnecessary to add tool enzymes, and the protein can be freed from the precursor protein after being induced under certain...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K14/00C12N15/63C12N1/21C12R1/19
Inventor 刘宏民张贵星王西新章旭耀王龙珍王志茹袁君郑一超赵文郑甲信
Owner ZHENGZHOU UNIV
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