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Long-acting single-chain insulin analog and conjugate thereof

A technology for single-chain insulin and analogs, which can be used in insulin, hormone peptides, animal/human proteins, etc., and can solve the problems of increased production process, complexity, and unsuitability for the preparation of single-chain insulin analogs.

Pending Publication Date: 2020-07-07
HANMI PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this approach is not suitable for the preparation of single-chain insulin analogues due to the trypsin recognition sequence present in proinsulin
As an alternative method, there is a method of removing methionine at the N-terminus of the protein using cyanogen bromide (CNBr) which has traditionally been widely used, but the extreme toxicity of CNBr, the increased complexity of the production process, the low yield rate etc. as the question has been raised

Method used

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  • Long-acting single-chain insulin analog and conjugate thereof
  • Long-acting single-chain insulin analog and conjugate thereof
  • Long-acting single-chain insulin analog and conjugate thereof

Examples

Experimental program
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Embodiment 1

[0270] Embodiment 1: Preparation of single-chain insulin analog expression vector

[0271] 1. Preparation of Proinsulin Analogs (A Chain-C Peptide-B Chain)

[0272] Expression vectors with nucleotide sequences were prepared to allow expression of insulin analogs and proinsulin analogs in E. coli. In order to release (free) the N-terminus of the insulin A chain, the expression vector includes a protein capable of expressing native proinsulin (B chain-C peptide-A chain; SEQ ID NO:4 and 5) and other proinsulin analogs (A chain-C Peptide-B chain, nucleotide sequence of SEQ ID NO: 6 and 7), and was prepared as follows.

[0273] [Table 1]

[0274]

[0275]

[0276] In order to prepare an expression vector encoding 86 amino acid proinsulin analogs, six primers were synthesized based on the reported proinsulin gene sequence (NM_000207.2, NCBI). In addition, using proinsulin cDNA (Origene) as a template, chain A, peptide C and chain B were respectively amplified by PCR, an...

Embodiment 2

[0308] Example 2: Expression of single chain insulin analogs

[0309] Under the control of the T7 promoter, the expression vector constructed in the above-mentioned Example 1 was used to express the recombinant single-chain insulin analogue. Escherichia coli BL21DE3 (E. coli B F-dcm ompT hsdS(rB-mB-)galλ(DE3); Novagen) was transformed with each recombinant single-chain insulin analog expression vector. For the transformation method, the method recommended by Novagen was used. Each transformed single colony transformed with each recombinant expression vector was collected, inoculated into 2X Luria Broth medium containing ampicillin (50 μg / mL), and incubated at 37° C. for 15 hours. Recombinant strain cultures were mixed with 2X LB medium containing 30% glycerol at a ratio of 1:1 (v / v), and 1 mL each was dispensed into cryo-tubes and stored at -140°C. The resultant was used as cell stock for production of recombinant fusion protein.

[0310]For expression of recombinant sing...

Embodiment 3

[0311] Example 3: Extraction and renaturation of recombinant single-chain insulin analogs

[0312] Cells from E. coli expressing the single-chain insulin analog obtained in the above examples were disrupted and renatured to convert the single-chain insulin analog into a soluble form. Cell pellets equivalent to 1L of culture were suspended in 1L of lysis buffer (20mM Tris-HCl pH 9.0, 1mM EDTA pH 8.0, 0.2M NaCl, 0.5% Triton X-100) ) machine disrupted the recombinant E. coli at 15,000 psi. After centrifugation at 12,000 g for 30 minutes, the supernatant was discarded, and the pellet was washed with 1 L of lysis buffer (50 mM Tris-HCl pH 9.0, 1 mM EDTA pH 9.0, 0.2M NaCl, 0.5% Triton X-100). After centrifugation under the same conditions as above, the supernatant was discarded, and the pellet was resuspended with distilled water. After centrifugation under the same conditions, washed E. coli inclusion body pellets were obtained. The washed inclusion body pellet was resuspended...

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Abstract

The present invention relates to a long-acting single-chain insulin analog, a conjugate thereof and uses of both. In addition, the present invention relates to a method for preparing a long-acting single-chain insulin analog and a conjugate thereof.

Description

technical field [0001] The present invention relates to long-acting single-chain insulin analogues, conjugates thereof, and uses thereof. Furthermore, the present invention relates to methods for the preparation of long-acting single-chain insulin analogs and conjugates thereof. Background technique [0002] Insulin is a polypeptide hormone composed of 51 amino acids secreted by the beta cells of the pancreas and is involved in the regulation of blood glucose in animals. Insulin consists of an A chain and a B chain linked by a disulfide bond, and the C-peptide is removed (hydrolyzed) by proteases in proinsulin in vivo to produce insulin from proinsulin. [0003] Insulin preparations are blood sugar regulators given to patients with type 1 diabetes who have a congenital defect in insulin production; secondary rises in blood sugar levels due to insulin resistance, insufficient insulin secretion, etc., and poorly controlled oral diabetes medications or oral Patients with type...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/64A61K47/60A61K47/68A61K47/54A61K38/28C07K14/62
CPCA61K47/60A61K47/68C07K14/62A61K47/6811A61K38/00A61K47/64A61K47/643A61K47/542A61K38/28A61K47/62
Inventor 许容豪吴宜林
Owner HANMI PHARMA
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