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Recombined long-acting glucagons peptide analogue and preparation method thereof

一种胰高血糖素、类似物的技术，应用在重组长效胰高血糖素样肽类似物及其制备领域，能够解决长半衰期、强生物活性等问题，达到延长半衰期、提高疗效、蛋白表达效率高的效果

Active Publication Date: 2013-08-28
NOVOPROTEIN SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But it is not a substrate of DIP-IV, so it has a longer half-life and stronger biological activity

Method used

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  • Recombined long-acting glucagons peptide analogue and preparation method thereof
  • Recombined long-acting glucagons peptide analogue and preparation method thereof
  • Recombined long-acting glucagons peptide analogue and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The construction of embodiment 1 genetically engineered bacteria

[0045] The exendin-4-HSA gene sequence was synthesized according to SEQ ID NO: 3, and the yeast codon was optimized. The methanolic yeast KEX2 restriction sequence KR and the xho1 restriction site were introduced into the N-terminal of the exendin-4 sequence, and the Not1 site was introduced into the C-terminal of the HAS. The synthesized fragment was double-digested with XhoI and NotI, and the vector pPICZαA plasmid was treated with XhoI and NotI double-digested at the same time, the fragments were recovered respectively, ligated and transformed into DH5α, and pPICZα-exendin-4-HSA transformants were screened. The correct pPICZα-exendin-4-HSA identified by sequencing was digested with sacI, electroporated to methanolic yeast X33, screened for zeocin resistance on YPD plate, and positive transformants were obtained. Pick a single colony, place it in a 250ml shake flask containing 25ml BMGY medium, and cu...

Embodiment 2

[0046] The cultivation and expression of embodiment 2 genetically engineered bacteria

[0047] 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 connected with 100 ul of the glycerol strain obtained in Example 1, and cultured on a shaker at 30° C. at 300 rpm for 24 hours. At this time, the bacterial concentration OD600 was between 6-20.

[0048] 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 O 0.93g / 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. Co...

Embodiment 3

[0049] Example 3 Separation and Purification of Genetically Engineered Bacteria Expression Products

[0050] Blue dye affinity chromatography: adjust the pH of the fermentation supernatant obtained in Example 2 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. Blue dye affinity chromatogram such as figure 2 shown

[0051] Hydrophobic chromatog...

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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 fusion protein, in particular to a recombinant long-acting glucagon-like peptide analog and a preparation method thereof. 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 o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/19C12P21/02C12R1/84
Inventor 朱化星蔡丽君王英明丁剑锋
Owner NOVOPROTEIN SCI INC
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