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Process for preparing and purifying Exendin-4 from colon bacillus

A technology of Escherichia coli and process, applied in the field of purification process of Exendin-4, can solve problems such as unsatisfactory yield and purity, achieve the effects of simple and easy operation steps, increase yield and purity, and simplify operation procedures

Inactive Publication Date: 2009-11-25
HENAN AGRICULTURAL UNIVERSITY
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Problems solved by technology

Two international biological companies, Qiagene and Invitrogen, have established purification processes for preparing target proteins from Escherichia coli, including bacterial culture, lysis, affinity chromatography purification of target proteins, and excising N-terminal fusion peptides with enterokinase. There are still deficiencies in the liquid formula, eluent formula and elution method, so the yield and purity of the target protein purified by this method, especially Exendin-4, are not satisfactory

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  • Process for preparing and purifying Exendin-4 from colon bacillus
  • Process for preparing and purifying Exendin-4 from colon bacillus

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Embodiment Construction

[0020] The present invention will be described in detail below in combination with specific embodiments.

[0021] 1. Preparation of reagents

[0022] (1) Configuration of lysis buffer 100ml

[0023] 50mMNaH 2 PO 4 0.690g NaH 2 PO 4 ·H 2 O (MW 137.99g / mol)

[0024] 300mM NaCl 1.754g NaCl (MW 58.44g / mol)

[0025] 10mM imidazole 0.068g imidazole (MW 68.08g / mol)

[0026] The pH was adjusted to 8.0 with NaOH.

[0027] Add now: Trition 100, 0.01%; Glycerol, 2%; β-mercaptoethanol, 10mM; PMSF, 0.1%; 100mM MgCl 2 , 1mM; 100mM MnCl 2 , 1mM; RNase, 1U; DNase, 1U

[0028] (2) Preparation of 100ml of washing buffer

[0029] 50mM NaH 2 PO 4 0.690g NaH 2 PO 4 ·H 2 O (MW 137.99g / mol)

[0030] 300mM NaCl 1.754g NaCl (MW 58.44g / mol)

[0031] 20mM imidazole 0.068g imidazole (MW 68.08g / mol)

[0032] The pH was adjusted to 8.0 with NaOH.

[0033] Add now: glycerol, 2%; β-mercaptoethanol, 10mM

[0034] (3) Preparation of 100ml of elution buffer

[0035] 50mM NaH 2 PO 4 0.6...

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Abstract

The invention relates to a process for preparing and purifying Exendin-4 from colon bacillus, belonging to the field of genetic engineering. The purified Exendin-4 is obtained by cultivating Exendin-4 high-yielding engineering bacterial strain through bacteria, carrying out non-denatured affinity chromatography, and eliminating N-end fusogenic peptide with enterokinase. Compared with the prior art, the invention improves the formulations of lysate and eluent and adopts a gradient elution technique, thereby enhancing the yield and the purity of a target protein; and besides, the invention simplifies the operating procedures, is simple and easy to operate in operating steps and is suitable for large-batch industrial production.

Description

technical field [0001] The invention relates to a purification process of Exendin-4, in particular to a process for preparing and purifying Exendin-4 from Escherichia coli. Background technique [0002] The key to the success of using genetic engineering methods to prepare economically valuable proteins is that one is to screen high-yield strains, and the other is to optimize the purification scheme to increase the purity and yield of the target protein. Both are indispensable. Especially, like Exendin-4, it is more difficult to purify small proteins with 39 amino acid residues and a molecular weight of only 4.2kD. Due to its extremely high economic value (7000 yuan / mg, a specific new drug for the treatment of type 2 diabetes) and its future use in industrialized production, the process of preparing and purifying Exendin-4 from Escherichia coli is extremely important because it determines Exendin-4 The yield, purity and feasibility of industrial production. Two internatio...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K1/14
Inventor 王小纯祖向阳陈红歌
Owner HENAN AGRICULTURAL UNIVERSITY
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