Expression and purification method of phospholipase A1 accessory protein PlaS

An auxiliary protein and expression method technology, applied in the field of molecular biology, to achieve the effect of relaxed experimental environment requirements, simple methods, and large protein adsorption capacity

Inactive Publication Date: 2018-05-08
ANHUI UNIVERSITY OF TECHNOLOGY AND SCIENCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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At present, there are few domestic and foreign research

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  • Expression and purification method of phospholipase A1 accessory protein PlaS
  • Expression and purification method of phospholipase A1 accessory protein PlaS
  • Expression and purification method of phospholipase A1 accessory protein PlaS

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Experimental program
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Effect test

Embodiment 1

[0031] Based on the bioinformatics analysis of the phospholipase A1 auxiliary protein plaS gene sequence (the analysis was provided by Detai Biotechnology (Nanjing) Co., Ltd.), the inventors found that there is a signal peptide at the front end, and the first 35AA is the signal peptide region ( figure 1 ),in:

[0032] figure 1 It is the analysis result of signal peptide of phospholipase A1 auxiliary protein plaS gene.

[0033] Therefore, the inventors used Primer 5.0 to design N-terminal truncated primers for plaS:

[0034] upstream primer P1CG GGATCC GAGATTTCACCGTTTG

[0035] downstream primer P2CCC AAGCTT CTGCTGCGCGTAGT

[0036] Among them, the underlined parts are the restriction sites of BamHI and HindIII respectively.

[0037] Specific steps are as follows:

[0038] Using the BL21 / SP28 previously constructed in our laboratory, that is, the plasmid of the recombinant plasmid plaS-pET-28 constructed in Chinese patent CN103333229, as a template, and P1 and P2 as pr...

Embodiment 2

[0052] The dSP28 bacterial strain successfully constructed in Example 1 is in LB medium, 37 DEG C, 200r / min cultured overnight, next day by 2 (V / V)% inoculum size seed liquid is inoculated to 100mL plus kanamycin (working Concentration of 50μg / mL) in the LB fermentation medium, until the concentration of bacteria to OD 600nm At about 0.7, add IPTG to induce 8h, prepare protein samples, and observe protein expression sites ( figure 2 ).

[0053] figure 2 It is the SDS-PAGE of P28 and dSP28, wherein, dSP28 is fermented with LB fermentation medium added with kanamycin and induced by IPTG.

[0054] Among them, 1, 3, 5, and 7 are the whole bacteria, fermentation broth supernatant, broken supernatant, and broken bacterial protein of P28 respectively, and 2, 4, 6, and 8 are the whole bacteria of dSP28, fermentation broth supernatant protein, broken supernatant protein, broken cell protein.

[0055] SDS-PAGE protein electrophoresis sample preparation for preliminary identificati...

Embodiment 3

[0085] The successfully constructed dSP28 strain in Example 1 was cultured overnight in LB seed solution, and the next day, the seed solution was inoculated into media of different components at an inoculum size of 2% (V / V) to induce expression of the target protein, and prepared protein samples, and observe the expression level of the target protein (Figure 3).

[0086] The different media are as follows:

[0087] a. TB culture medium;

[0088] b. TB medium plus 0.1wt% SDS;

[0089] c. TB medium plus 0.5wt% Tween 20;

[0090] d. TB medium plus 0.98wt% glycerol and 0.75wt% glycine;

[0091] e. TB medium plus 3wt% glycerol;

[0092] f. Lactose self-induction medium;

[0093] g. Add 0.75wt% glycine 6h after self-induction of lactose.

[0094] The above-mentioned five kinds of fermentation media a, b, c, d, and e were inserted into the seed liquid, and then cultivated at 37°C and 200r / min for 2h, that is, OD 600nm At about 0.7, add 0.2mM IPTG, and ferment for 22 hours at 2...

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Abstract

The invention provides an expression method of phospholipase A1 accessory protein PlaS. The expression method comprises the following steps: removing signal peptide from the end N of a phospholipase A1 accessory protein PlaS gene to obtain truncated accessory protein, then constructing an escherichia coli/truncated accessory protein expression strain, and inoculating the expression strain into a culture medium to perform target protein inducible expression. The inventor adopts an end-N truncating technology to truncate the end N of the plaS gene by 35AA, and successfully constructs the BL21/dSP28 expression strain, so that a large amount of the accessory protein can be expressed. Furthermore, through optimization of components for expressing a culture medium, the culture medium which is capable of realizing soluble expression of a large amount of target protein and is easy to purify is obtained. The target protein is purified by adopting different purification modes, and an optimal purification method is determined. The culture medium enables a large amount of the target protein to be solubly expressed in broken supernatant, so that tedious operation of denaturization and renaturation in a protein purification process is reduced; the purification method increases the protein yield, so that the purification process is simpler and more convenient.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for expressing and purifying phospholipase A1 auxiliary protein PlaS. Background technique [0002] Phospholipase A1 is an important class of hydrolytic enzymes in organisms. It hydrolyzes phospholipids to generate lysophospholipids and free fatty acids. It has been widely used in industry, mainly in plant degumming, food industry, pharmaceutical industry and so on. There is also a protein in phospholipase A1 in Serratia marcescens that is involved in the activity, secretion and growth of phospholipase A1. This protein is called the auxiliary protein PlaS of phospholipase A1. Su Yannan of Anhui Engineering University and others found in the process of studying the production of phospholipase A1 by Serratia marcescens: there is an auxiliary protein coding sequence plaS downstream of the phospholipase A1 coding gene plaA, which is related to the high ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K14/00C07K1/14
CPCC07K14/00C12N15/70
Inventor 薛正莲朱昊王洲刘艳陈阿娜杨蒙甘玉飞
Owner ANHUI UNIVERSITY OF TECHNOLOGY AND SCIENCE
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