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Preparation method of recombinant human glucagon-like peptide-1 analogue fusion protein

A glucagon and fusion protein technology, which is applied in the field of preparation of recombinant human glucagon-like peptide-1 analog fusion protein, can solve the problem of easy degradation of the target protein, low recovery rate of effective activity, and instability of fusion protein products And other issues

Active Publication Date: 2020-10-23
MABWELL (SHANGHAI) BIOSCIENCE CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the technical problems that the target protein is easy to degrade, the recovery rate of effective activity is low, and the fusion protein product is unstable during the production process of the glucagon-like peptide-1 (GLP-1) analog fusion protein, the present invention optimizes the recombinant human glucagon in the upstream On the basis of molecular design of glucagon-like peptide-1 analog fusion protein, systematic analysis and research on downstream production processes such as recombinant bacterial fermentation, induced expression of target protein, product separation and purification

Method used

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  • Preparation method of recombinant human glucagon-like peptide-1 analogue fusion protein
  • Preparation method of recombinant human glucagon-like peptide-1 analogue fusion protein
  • Preparation method of recombinant human glucagon-like peptide-1 analogue fusion protein

Examples

Experimental program
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Effect test

Embodiment 1

[0054] Example 1: Construction of Engineering Bacteria Expressing Recombinant Human Glucagon-Like Peptide-1 Analogue Fusion Protein

[0055] According to the method of CN201310331182.6 invention patent application, the GLP-1 analog fusion protein expression engineering bacteria were prepared. In short, the N-terminal part of the GLP-1 analog-E3-HSA gene fragment was artificially synthesized, the HSA coding gene sequence was obtained by PCR amplification, the GLP-1 analog-E3-HSA fusion gene was obtained by overlapping PCR, and its sequence was identified by sequencing As shown in SEQ ID NO:5. The GLP-1 analogue-E3-HSA / pPIC9 recombinant plasmid was obtained by XhoI and NotI double digestion and introduced into the pPIC9 plasmid. The GLP-1 analogue-E3-HSA / pPIC9 recombinant plasmid was linearized with SalI and then electrotransformed into Pichia pastoris GS115 competent cells, screened and identified, and positive transformants were obtained, which were induced and expressed on a...

Embodiment 2

[0056] Example 2: Large-scale fermentation culture and induced expression method of engineering bacteria expressing recombinant human glucagon-like peptide-1 analogue fusion protein

[0057] 1. Preparation of medium

[0058] Weigh 46.5g of calcium sulfate dihydrate, 910.0g of potassium sulfate, 383.0g of magnesium sulfate, 4000.0g of glycerin, measure 1335.0ml of 85% phosphoric acid and add it to a stainless steel bucket, add water to dissolve it, add 207.0g KOH and 20.0ml GPE defoamer to dissolve Afterwards, add purified water to a fixed position in a stainless steel bucket to make a constant volume of 90L.

[0059] 2. Sterilization

[0060] Add the prepared medium into the fermenter with a peristaltic pump, and carry out actual sterilization. The sterilization conditions are 115°C and 30min. After the fermenter is sterilized, the feeding pipeline and transplanting pipeline are sterilized online for 20 minutes with branch steam.

[0061] 3. Preparation before transplantati...

Embodiment 3

[0074] Example 3: Purification method of recombinant human glucagon-like peptide-1 analogue fusion protein

[0075] 1. Affinity BlUE chromatography

[0076] Packing: packing Blue Sepharose Fast Flow 3.1L, column height 10cm, column BPG 200 / 500.

[0077] Solution: Balance solution BUFFER A (10mmol / L NaAc-HAc pH5.0+5mmol / L EDTA-2Na+0.15mol / L NaCl).

[0078] Washing solution: BUFFER B1 (25% ethylene glycol).

[0079] Eluent: BUFFER B2 (20mmol / L Tris-HCl pH8.0+1mol / L NaCl+25% ethylene glycol+5mmol / L EDTA-2Na).

[0080] First use the balance solution BUFFER A to balance the chromatography column for 2-3CV (column volume), and keep the flow rate at 50-80cm / h throughout the chromatography process. After equilibration, start to load the sample. The load of the sample should be controlled at 6-10mg / ml. After the sample is loaded, first equilibrate the column with 2-2.5CV of BUFFER A, then wash the column with 2-2.5CV of BUFFER B1, and then use 2 ~ 2.5CV of BUFFERB2 elution, when th...

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Abstract

The invention provides a preparation method of recombinant human glucagon-like peptide-1 analogue fusion protein. By using an engineered yeast fermentation broth supernatant containing the recombinanthuman glucagon-like peptide-1 analogue fusion protein as an object, heating enzyme deactivation is carried out in the presence of sodium octoate and tranexamic acid, so that the degradation and biological activity loss of target protein in a separation and purification process are reduced, and the stability of the recombinant human glucagon-like peptide-1 analogue fusion protein for injection isimproved.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to a separation and purification method for recombinantly expressing an exogenous target protein by genetically engineered bacteria, and more specifically to a preparation method for a recombinant human glucagon-like peptide-1 analogue fusion protein. Background technique [0002] Glucagon-like peptide-1 (GLP-1) and its analogs such as Exendin-4 are widely used to study the treatment of type 2 diabetes, because the GLP-1 polypeptide is digested by protease dipeptidyl peptidase Ⅳ (DPP- IV) is rapidly inactivated, and its half-life in plasma is very short, making it difficult to be widely used clinically; while Exendin-4 is insensitive to the enzymatic degradation of DPP-IV, its stability increases, but its molecular weight is low (4187.61 D), short half-life in vivo, so it must be injected twice a day, which hinders clinical use. Many efforts have been made to solve this technical ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/81C12P21/02C07K1/18C07K1/22C07K1/20C07K1/16C12R1/84
CPCC07K14/605C07K2319/31C12N15/815C12P21/02
Inventor 任杰游猛梅菲方鹏谭小钉陆游范敏曹雨霞
Owner MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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