Separation and purification method of c-di-GMP (cyclic diguanylate)

A c-di-gmp, separation and purification technology, applied in the field of separation and purification of cyclic guanosine diphosphate, can solve the problems of reducing the service life of chromatographic columns, harm to operators and environment, complicated separation and purification processes, etc., and achieves reduction of separation and purification. Time, production cost reduction, effect of simplified steps

Inactive Publication Date: 2014-03-05
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] It can be seen that the separation and purification process of the traditional method is complicated, time-consuming and energy-consuming.
Reversed-phase chromatographic columns have stricter requirements on samples. Impurities (such as enzymes) that may be deposited on the column must be removed first, otherwise the service life of the chromatographic column will be reduced; the lyophilization process requires a lyophilizer and consumes a lot of time, resulting in Low production efficiency
And because a large amount of organic solvents are used in the preparation process, it will cause harm to the operator and the environment, and the environmental protection effect is poor

Method used

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  • Separation and purification method of c-di-GMP (cyclic diguanylate)

Examples

Experimental program
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Effect test

Embodiment 1c

[0027] The preparation (enzymatic reaction method) of the crude product solution of embodiment 1c-di-GMP

[0028] GTP (purchased from Sigma) and the enzyme VCA0956 with c-di-GMP synthesis activity were added to the reaction buffer (75mM Tris-HCl pH 8.0, 250mM NaCl, 25mM KCl, 10mM MgCl 2 ), react overnight at room temperature to obtain a crude product solution containing c-di-GMP.

[0029] Among them: For the detailed methods and steps of the expression and purification of the above-mentioned VCA0956 and the preparation of the c-di-GMP crude product solution, please refer to the literature: Rita Tamayo, Anna D. Tischler, and Andrew Camilli. The EAL domain protein VieA is a cyclic diguanylate phosphodiesterase. J. Biol. Chem., 2005, 280, 33324-33330.

Embodiment 2

[0031] (1) Take 4 mL of the crude product solution containing c-di-GMP prepared in Example 1, and load it on the ion-exchange chromatographic column GE AKTA FPLC equipped with Mini Q 4.6 / 50PE at a flow rate of 1 mL / min;

[0032] (2) After loading the sample, use 25mM Tris-HCl (pH8.0) (A) and 25mM Tris-HCl (pH8.0) + 1M NaCl (B) gradient elution at a flow rate of 1.5mL / min, gradient elution The stripping is carried out at 0→20 times the column volume, the concentration value of eluent A is carried out at 100%→0, and the concentration value of eluent B is carried out at 0→100%;

[0033](3) Use a UV detector to detect at 254nm; the peak appears when the conductivity of the eluent is 35.97mS / cm, collect the eluate at the position of the peak, and confirm its structure with mass spectrometry [(ESI+): m / e(relative intensity ): [M+1] + 691.2, [M+2]2+346.2], this part of the eluate was lyophilized to obtain solid c-di-GMP (results such as figure 1 shown);

[0034] (4) The product of...

Embodiment 3

[0036] (1) Get 30 mL of the crude product solution containing c-di-GMP prepared in Example 1, and load it on a GE AKTA FPLC equipped with a Source Q HP 10 / 10 ion-exchange chromatographic column at a flow rate of 3 mL / min;

[0037] (2) After loading the sample, use 25mM Tris-HCl (pH8.0) (A) and 25mM Tris-HCl (pH8.0) + 1M NaCl (B) gradient elution at a flow rate of 3mL / min, gradient elution Carry out at 0→20 times the column volume, the concentration value of eluent A is carried out at 100%→0, and the concentration value of eluent B is carried out at 0→100%;

[0038] (3) Detect at 254nm with an ultraviolet detector; when the eluent conductance is 36.03mS / cm, the peak is collected, and the eluent at the peak position is collected, and its structure is confirmed by mass spectrometry, and this part of the eluent is freeze-dried to obtain final product Solid c-di-GMP;

[0039] (4) The product of step (3) is detected by reverse-phase HPLC, and the high-performance liquid chromatogra...

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Abstract

The invention discloses a separation and purification method of c-di-GMP (cyclic diguanylate), comprising the following steps of: directly sampling a c-di-GMP crude product solution on a fast protein liquid chromatography equipped with an ion exchange chromatographic column; carrying out gradient elution by using a buffer solution with pH of 8.0; carrying out detection under 254 nm by an ultraviolet detector; collecting an eluent in the peak position; confirming a structure by using a mass spectrum; and freeze-drying the eluent with target compounds, thereby obtaining the solid c-di-GMP. The c-di-GMP prepared according to the method provided by the invention has the purity of over 95% through reversed-phase high performance liquid chromatography detection, and realizes c-di-GMP separation and purification from the c-di-GMP crude product solution in one step; moreover, the method provided by the invention has simple technology, short period and nonuse of toxic organic solvents, and is a novel method with high efficiency, environment friendliness and suitability for industrialized production.

Description

technical field [0001] The present invention relates to a kind of separation and purification method of cyclic guanosine diphosphate (Cyclic diguanylate, c-di-GMP), specifically, relate to the fast protein liquid chromatography (Fast protein liquid chromatography, FPLC) method for separating and purifying c-di-GMP. Background technique [0002] Cyclic diguanylate (c-di-GMP) is a novel second messenger with bacterial specificity, which widely exists in various eubacteria, but not in eukaryotes. c-di-GMP participates in the regulation of various physiological processes of bacteria, including the formation of biofilm, the regulation of motility, and the expression of virulence factors. Biofilm, as the main mode of survival of bacteria in nature, has brought serious harm to human production and life. In addition to endangering the environment and industrial production, according to the NIH survey report, more than 80% of bacterial infections are related to biofilms, which can ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07H19/213C07H1/06
Inventor 谷立川朱春原许素娟陈颖李冰清
Owner SHANDONG UNIV
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