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A sequence that can be modified by 4' phosphopantethein and its method for immobilizing proteins

A technology of phosphopanthenylmercaptoethylamine and acyl carrier protein, which is applied in the field of immobilizing proteins, can solve the problems of unfavorable macromolecular substrate and product diffusion, limiting the wide application of immobilized proteins, and loss of protein (enzyme) activity, etc. Market value and application prospects, the effect of reducing inclusion body formation and high activity

Active Publication Date: 2017-11-03
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

At present, the various enzyme immobilization methods mentioned above have their own shortcomings. The molecular size selectivity of the polymer gel or semi-permeable membrane in the embedding method is not conducive to the diffusion of macromolecular substrates and products. The activity of the protein (enzyme) is greatly lost due to the reaction, and the immobilized protein (enzyme) in the adsorption method is easily affected by the pH and ionic strength of the reaction medium and falls off from the carrier.
The covalent binding method presents good stability and reusability due to the covalent binding between protein (enzyme) molecules and carriers. It is currently the most active type of protein (enzyme) immobilization method. However, non-directional covalent Valence immobilization Due to the uneven distribution of protein (enzyme) on the surface of solid media, it often leads to a large loss of protein (enzyme) activity
The currently developed protein (enzyme) directional immobilization methods, such as using the specific interaction between the protein (enzyme) and the corresponding antibody, the specific interaction between the protein (enzyme) and the corresponding ligand, etc., have complicated operation procedures and low efficiency. These deficiencies limit the wide application of immobilized proteins (enzymes), and become the main problems to be solved urgently.
At present, the existing protein (enzyme) immobilization technology has complicated operation process and lacks specificity

Method used

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  • A sequence that can be modified by 4' phosphopantethein and its method for immobilizing proteins
  • A sequence that can be modified by 4' phosphopantethein and its method for immobilizing proteins
  • A sequence that can be modified by 4' phosphopantethein and its method for immobilizing proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Cloning of an Escherichia coli acyl carrier protein (ACP) gene.

[0022] (1) Extraction of Escherichia coli total DNA

[0023] 1) Pick a single colony of Escherichia coli MG1655 in LB liquid medium and culture overnight at 37°C.

[0024] 2) Centrifuge at a centrifugal force of 5000g for 10 min, collect the bacteria, and obtain the genomic DNA of Escherichia coli by referring to the DNA extraction instructions of TIANGEN Company.

[0025] (2) Cloning of ACP gene

[0026] 1) Design 2 primers:

[0027]

[0028] 2) A fusion ACP gene (SEQ ID NO: 1) was obtained by amplifying with F1 and R1 primers, and the obtained fusion ACP gene fragment was digested with Nco I and EcoR I respectively, and connected to the pET28b vector.

[0029] (3) Fusion expression of ACP and green fluorescent protein (GFP)

[0030] 1) Construction of expression vector

[0031] The GFP gene was excised from the intermediate vector pET30a-GFP with EcoR I and Hind III, connected to the e...

Embodiment 2

[0034] Example 2 Cloning of a short peptide gene that can be modified by 4'-phosphopantethein and its fusion expression with the GFP gene.

[0035] (1) Cloning of short peptide genes that can be modified by 4'-phosphopantetheine

[0036] 1) Design 3 primers:

[0037]

[0038] 2) Using the GFP gene as a template, use primers F2, F3, and R2 to amplify to obtain a fusion gene S-GFP (the fusion gene S-GFP includes a gene that can be introduced by primers F2, F3, and R2 and can be introduced by 4' phosphopanthene Mercaptoethylamine-modified sequence (SEQ ID NO: 2). Primers R2, F3, and F2 were successively used as up primers in the first, second, and third rounds of PCR for PCR amplification.

[0039] The obtained fusion gene S-GFP fragment was digested with Hind III and EcoR I, and connected to pET28b vector.

[0040] (2) Expression of fused GFP gene (S-GFP)

[0041] 1) Construction of expression vector

[0042] The fused GFP gene (S-GFP) was digested with EcoR I and Hind III...

Embodiment 3

[0045] Example 3 Immobilization of the target protein GFP

[0046] (1) Immobilization of fusion protein ACP-GFP

[0047] 10ml of Escherichia coli culture expressing the fusion protein ACP-GFP, the cells collected by centrifugation were ultrasonically disrupted in 4ml of buffer solution (50mM NaH2PO4, 0.3M NaCl, 20mM imidazole, pH 8.0), and purified by Ni column affinity chromatography , the obtained high-purity fusion protein was dialyzed, and the purified fusion protein was modified with 4' phosphopantethein with the enzyme AcpS, and then dialyzed into immobilization buffer (50mmol Tris HCl, 5mmol EDTA-Na, pH 8.0), To measure the protein concentration, mix about 1mg of recombinant protein with 0.2ml SulfoLink coupling resin (Thermo Scientific), incubate at 30°C for 1h, measure the change of recombinant protein concentration before and after immobilization, calculate the immobilized amount of recombinant protein, and use 50mML-cysteine· The column was washed 3 times with HCl ...

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Abstract

The invention discloses a sequence capable of being modified by 4'-phosphopantetheine and a method thereof for immobilizing protein, belonging to the technical field of bioengineering. The sequence capable of being modified by 4'-phosphopantetheine is an Escherichia coli acyl carrier protein gene sequence disclosed as nucleotide sequence SEQ ID NO:1 or oligopeptide capable of being modified by 4'-phosphopantetheine disclosed as nucleotide sequence SEQ ID NO:2. The method comprises the following steps: obtaining a fusion protein comprising the target protein by using the Escherichia coli recombinant expression system constructed by using the expression vector comprising the sequence, carrying out 4'-phosphopantetheine modification by using enzyme AcpS or Sfp as a catalyst, and carrying out immobilization to immobilize the target protein. The immobilization method has the advantages of mild reaction conditions and high immobilization efficiency, and is very simple to operate; the immobilized target protein is stable and has very high activity; and thus, the method has great market value and application prospects.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and particularly relates to a sequence that can be modified by 4'-phosphopantethein and a method for immobilizing proteins thereof. Background technique [0002] Immobilized protein (enzyme) has a series of advantages such as high storage stability, easy separation and recovery, repeated use, continuous and controllable operation, simple process, etc., and because it has the ecological environment of saving resources and energy, reducing or preventing pollution Effects meet the strategic requirements of sustainable development, and have a wide range of applications in the fields of biomedicine, food, bioanalysis and basic research in biology. The traditional protein (enzyme) immobilization methods include embedding method, cross-linking method, adsorption method and covalent binding method to realize the immobilization of enzyme. The preparation process of the embedding method is simple a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/11C12N15/70C12N15/62C12N1/21C07K17/00C12N11/00
Inventor 王声斌汪华珍孙长胜袁启航陈长超洪福林马皖燕
Owner SOUTH CHINA AGRI UNIV
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