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Preparation method of insulin

A technology of insulin and trypsin, applied in the direction of fermentation, etc., can solve the problems of impracticality in large-scale production

Inactive Publication Date: 2014-08-13
VALIN TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are not practical for large-scale production

Method used

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  • Preparation method of insulin
  • Preparation method of insulin
  • Preparation method of insulin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Ways to Produce Insulin Efficiently

[0092] expression cloning

[0093] pass software to optimize the nucleotide sequence encoding the insulin precursor protein for improved expression in Escherichia coli. Synthetic genes were then assembled from the synthetic oligonucleotides and cloned into pET24a (Novagen) using NdeI and BlpI restriction enzyme sites. Escherichia coli BL21 strain (DE3) cells were transfected with the above clones. The gene is transcribed under the control of the T7 promoter and lac operator. Transcription termination is controlled by the T7 terminator and translation initiation is initiated by the pET24 ribosome binding site. Expression of the 12.7 kD fusion protein serving as an insulin precursor was induced by IPTG, resulting in very high levels of insulin precursor. This precursor contains a Cu / Zn SOD moiety as the amino-terminal moiety fused to a proinsulin-like moiety via an Arg residue, where Lys-Arg links the B and A chains of insulin. ...

Embodiment 2

[0106] Efficiently produced insulin is active in rats

[0107] In order to evaluate the potential therapeutic effect of the long-acting insulin analog produced in Example 1, in streptozotocin (STZ)-induced diabetic rats by measuring their blood glucose levels at different time points after administration, test Insulin - LysArg and Lantus (Junod et al., 1969). Young Sprague-Dawley male rats, approximately 9 weeks of age, received a single intraperitoneal injection of 60 mg STZ / kg body weight. Five days after induction of diabetes, most animals exhibited blood glucose values ​​above 300 mg / dL. The animals received the test material equivalent to 6 IU / kg by subcutaneous injection on 3 consecutive days. The study consisted of 3 experimental groups of 6 animals each, treated with the 2 different insulins specified above and vehicle as control. Blood glucose was measured before treatment and 1, 4, 8, and 24 hours after each dosing day. Such as Figure 5 As shown, both test mate...

Embodiment 3

[0109] Efficiently produced insulin with long-acting insulin activity in dogs

[0110] The effect of different insulin preparations on lowering blood glucose levels was tested in alloxan-induced diabetic beagle dogs. Eight healthy castrated female dogs, aged 2–5 years and weighing 10–17 kg, were injected intravenously with 50 mg / kg body weight of alloxan. Three days after treatment, all dogs developed a reproducible experimental diabetic state, showing hyperglycemia and other characteristic symptoms (Watanabe et al., The pathological features of alloxan diabetes in beagle dogs, J Toxicol Pathol, 2004, 17, 187- 195).

[0111] Insulin-LysArg ("Insulin KR") prepared as described above was compared with two commercially available insulin formulations, Humulin R (Elli Lily) and Lantus (Sanofi-Aventis). The three formulations and the vehicle control were tested in a randomized crossover study in 8 diabetic beagle dogs.

[0112] Different insulin preparations and the control were ...

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Abstract

The invention provides a preparation method of insulin, relates to a method for transforming an insulin precursor into an active insulin compound, preferably a method for improving long-acting insulin; and particularly provides a method, wherein lysine residues in Lys-Arg precursor insulin can be reversibly closed, and parenzyme can accurately cut the reversibly closed Lys-Arg precursor insulin.

Description

technical field [0001] The present invention relates to improved methods of converting insulin precursors into long-acting active insulin compounds. Background technique [0002] Insulin is a pancreatic hormone involved in the regulation of blood sugar concentration. For example, human, porcine, and bovine insulin, insulin analogs, and mixed insulins are administered to insulin-dependent diabetics to regulate their blood glucose concentrations. [0003] Mature insulin is a two-chain peptide hormone containing 51 amino acids. Its A-chain consists of 21 amino acids and its B-chain consists of 30 amino acids. The molecule contains 2 interchain disulfide bonds and 1 A-chain intrachain disulfide bond. In the body, the hormone is first synthesized as a long precursor molecule, preproinsulin, which is subsequently processed into proinsulin and insulin. The initial processing step is the proteolytic removal of the propeptide during the transfer of the nascent chain across the en...

Claims

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Application Information

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IPC IPC(8): C12P21/06C07K1/20C07K1/18C07K1/14
Inventor 雅各布·哈特曼江立新
Owner VALIN TECH
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