Preparation of recombinant long-acting glucagon peptide analogue

A technology for glucagon and analogues, which is applied in the field of preparation of recombinant long-acting glucagon-like peptide analogues, which can solve the problems of strong biological activity and long half-life, so as to improve curative effect, prolong half-life, and improve protein expression The effect of efficiency and purification product yield

Inactive Publication Date: 2009-02-25
SHANGHAI SINOBIO BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Preparation of recombinant long-acting glucagon peptide analogue
  • Preparation of recombinant long-acting glucagon peptide analogue
  • Preparation of recombinant long-acting glucagon peptide analogue

Examples

Experimental program
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Embodiment 1

[0035] Embodiment 1 The cultivation and expression of genetically engineered bacteria

[0036] Shake flask strain culture: 5 Erlenmeyer flasks with a capacity of 1L, each containing 200ml of YPD medium, the medium formula is 10g of yeast extract (1%), 20g of peptone (2%), and 20g of glucose (2%). Dissolve in deionized water and make up to 1L, steam sterilize at 115°C for 20 minutes. Each bottle was inoculated with 100 ul of glycerol strains, and cultured on a shaker at 30° C. at 300 rpm for 24 hours. At this time, the OD600 of the bacterial concentration was between 6 and 20.

[0037] 30L tank fermentation culture: 30L automatic fermentation tank (Biostat c-dcu, Sartourius), specific operation can refer to its instruction manual. 15L of basal salt medium is filled in a 30L tank, and the formula is CaSO 4 2H 2 O0.93g / l, K 2 SO 4 18.2g / l, MgSO 4 7H2O, KOH 4.13g / l, H 3 PO 4 26.7ml / l, 40g / l glycerin were dissolved in deionized water and adjusted to 15L. Connect the pH el...

Embodiment 2

[0038] Example 2 Separation and Purification of Genetically Engineered Bacteria Expression Products

[0039] Blue dye affinity chromatography: adjust the pH of the fermentation supernatant obtained in Example 1 to 7.0 with 6M NaOH, and load it into blue dye affinity chromatography equilibrated with 20 mM phosphate at pH 7.0 and 500 mM NaCl buffer The column has a column bed diameter of 100 mm, a column bed height of 15 cm, and a column bed volume of 1000 ml. The flow rate is 8L / hour, and the sample is loaded after 3 hours. After loading the sample, first wash off unbound substances with 20 mM phosphate buffer at pH 7.0 and 128 mM KSCN buffer, and then wash off the fusion protein bound on the column with 20 mM phosphate buffer at pH 7.0 and 230 mM KSCN buffer. The protein peak was collected for detection by ultraviolet absorption at 280nM, and a total of 3200ml of the eluate was collected.

[0040] Hydrophobic chromatography: Add 630ml of 3M ammonium sulfate solution to the c...

Embodiment 3

[0042] The oral glucose tolerance test of embodiment 3 mice

[0043] Kunming mice aged 8-10 weeks were used, and they were adaptively fed for 1 week after purchase, with free access to food and water. Fasting for 16-18 hours one day before the test, intravenously inject 5 mg / Kg of human serum albumin or the recombinant glucagon-like peptide analog albumin fusion protein injection formulated above according to body weight. One hour after the administration, 1.5 mg / g of body weight glucose was administered by oral infusion, and blood was taken from the tail vein 10 minutes before and 10, 20, 30, 60, and 120 minutes after the administration of glucose, and the glucose level was measured.

[0044] The result is as follows:

[0045] time HSA group (n=6) mM / L E4 / HSA group (n=6) mM / L 10 minutes before sugar 4.2 3.0 10 minutes after giving sugar 9.8 6.3 20 minutes 10.2 6.5 30 minutes 9.6 6.1 60 minutes 6.2 4.3 120 minutes 5.5 3.8...

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Abstract

The invention relates to recombinant proteins and discloses a preparation method of a long-acting glucagon peptide analog, including the following steps: inoculating gene engineering Pichia pastoris strain in a fermentation tank; performing fermentation culture in the fermentation tank; centrifuging fermentation liquor and collecting the supernatant; separating and purifying the long-acting human glucagon peptide analog in the supernatant of the fermentation liquor so as to finally obtain a product with a purity degree of greater than 95%. The preparation method of the long-acting glucagon peptide analog has easy control for technologic conditions, thereby being capable of obtaining the long-acting glucagon peptide analog with biological activity, and having high protein expression efficiency and high product purification yield.

Description

technical field [0001] The invention relates to a recombinant protein, in particular to a preparation method of a recombinant long-acting glucagon-like peptide analogue. Background technique [0002] Diabetes is a metabolic disease with multiple etiologies and has become a major disease that seriously endangers human health and life in modern society. According to the data provided by the World Health Organization (WHO), the prevalence of diabetes in developed countries has reached as high as 5%-10%, and in my country it is about 3%. Among them, the number of type 2 diabetes patients currently accounts for more than 90% of all diabetes patients. [0003] Glucagon-like peptide-1 (glucagon.like peptide.1GLP-1) can bind to the GLP-1 receptor and control blood sugar through multiple actions, including stimulating insulin secretion, simultaneously inhibiting glucagon and gastric emptying , but will not produce hypoglycemia, and has a good effect on the treatment of type II diab...

Claims

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Application Information

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IPC IPC(8): C12P21/06C07K1/22C07K1/20C07K1/18C07K1/14C12R1/84
Inventor 朱化星蔡丽君王英明丁剑锋
Owner SHANGHAI SINOBIO BIOTECH
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