A kind of preparation method of mettriptine

A technology for mettriptin and its purpose, which is applied in the field of preparation of mettriptin, can solve the problems of low yield and difficult industrial scale-up, and achieve high yield, high renaturation efficiency, and easy industrial scale-up Effect

Active Publication Date: 2022-05-10
WUHAN HITECK BIOLOGICAL PHARMA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems existing in the prior art that mettriptin often exists in the form of inclusion bodies in the bioengineering preparation process, undergoes complex denaturation and renaturation processes, is difficult to scale up for industrialization, and has a low yield, the present invention provides A new method for preparing mettriptin. The overall technical plan is to express mettriptin in a prokaryotic expression system to obtain inclusion bodies, dissolve the inclusion bodies in a milder way, and perform renaturation by hydrophobic chromatography

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  • A kind of preparation method of mettriptine
  • A kind of preparation method of mettriptine
  • A kind of preparation method of mettriptine

Examples

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Embodiment 1

[0048] Example 1: Construction of Metreptine Recombinant Expression Vector

[0049] 1.1 Sequence optimization and synthesis of mettriptin expression gene

[0050] Referring to the prokaryotic Escherichia coli codon usage frequency table, combined with factors such as mRNA secondary structure stability and GC content balance, bases were optimized without changing the amino acid sequence of metreptine shown in SEQ ID NO.1 In order to maximize the expression of the target protein, the optimized mettriptin expression gene sequence is shown in SEQ ID NO.2. The sequence was synthesized by chemical synthesis.

[0051] 1.2 Construction of mettriptin recombinant expression vector

[0052] The prokaryotic expression vector was used to construct the recombinant expression vector of mettriptin. In this example, the recombinant expression vectors were all constructed by the method of seamless cloning. The target protein (metriptin) genes are all immediately after the start codon ATG, a...

Embodiment 2

[0087] Example 2: Construction and Induced Expression of Metreptine Genetic Engineering Bacteria

[0088] The recombinant expression vector constructed in Example 1.2 was passed through CaCl 2 Heat shock transformation method or electroporation method (Electroporation) is transferred to the adapted Escherichia coli expression strain cells, and the recombinants are obtained after screening, which is the genetic engineering strain. Listed below to illustrate:

[0089] 2.1 Construction and induced expression of pET-DE3 engineering strain

[0090] The pET32a(+)-leptin recombinant expression vector constructed in Example 1.2.1 was transformed into BL21(DE3) Escherichia coli expression strain, and cultured on LB solid medium containing ampicillin with a final concentration of 100 μg / ml at 37°C for 13~ After 16 hours of screening, the recombinant was obtained, which was the mettriptin genetically engineered strain, and was denoted as the pET-DE3 engineered strain.

[0091] (1) Ind...

Embodiment 3

[0103] Example 3: Small-scale fermentation of pET-DE3 engineering bacteria and preparation and washing of inclusion bodies

[0104] Get the pET-DE3 engineering bacterium that freezes the embodiment 2.1 that constructs, after activation, expanded culture, inoculate in 5L fermentation medium by 10% inoculum size (fermentation medium composition is: glucose 5.0g / L, peptone 6.0g / L L, yeast powder 12.0g / L, NaCl1.0g / L, NH 4 Cl 2g / L, Na 2 HPO 4 12H 2 O 30.0g / L, KH 2 PO 4 3.0g / L, MgSO 4 ·7H 2 O 1.0g / L) in the fermentation culture (temperature 37 ℃, rotating speed 200rpm, ventilation rate 1.8Nm 3 / h, tank pressure 0.2bar); control the dissolved oxygen above 30% in the early stage of fermentation, gradually adjust the speed to 600rpm when it is insufficient, and then adjust the ventilation to 2.5Nm 3 / h, and finally fed with carbon source (glucose 600g / L) and nitrogen source (peptone 150g / L, yeast powder 150g / L) to control dissolved oxygen at about 30%; in the early stage of fe...

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Abstract

The invention discloses a method for preparing mettriptin, specifically, the mettriptin gene is expressed through a prokaryotic expression system to obtain inclusion bodies, the inclusion bodies are dissolved, and hydrophobic chromatography renaturation is performed to obtain mettriptin protein. Both inclusion body solubilization and hydrophobic chromatographic renaturation are carried out in a gentle manner, and antioxidants and / or metal chelating agents can also be added during the process to inhibit the oxidation and degradation of the target protein. The method for preparing mettriptin is easy to realize industrial scale-up, high renaturation efficiency, high yield and good biological activity.

Description

technical field [0001] The invention relates to the technical field of protein engineering, in particular to a preparation method of mettriptin. Background technique [0002] Metreptin is a recombinant human leptin derivative (rmetHuLeptin) expressed in the E. coli expression system. It contains 147 amino acid residues. Compared with natural human leptin protein, its N-terminus has one more methionine residue. base, so it is also called recombinant human methionyl leptin protein. Metreptine has no glycosylation modification, its 97th cysteine ​​residue and 147th cysteine ​​residue form an intra-chain disulfide bond to block its C-terminus, and its relative molecular weight is about 16.15k Da . On February 24, 2014, the U.S. FDA approved the marketing of Metreptine preparations (trade name: Myalept, product of Amylin Company), which are used as supplementary therapy for the treatment of leptin deficiency complications in patients with congenital or acquired lipodystrophy. T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/575C07K1/20C07K1/14C12N15/16C12N15/70
CPCC07K14/5759C12N15/70
Inventor 汤华东梅芸董瑶童齐金陈亚
Owner WUHAN HITECK BIOLOGICAL PHARMA
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