Preparation method of metreleptin

A technology of metriptin and equilibration solution, applied in the field of protein engineering, can solve the problems of difficult industrialization and amplification, low yield, etc., and achieve the effects of high renaturation efficiency, high yield, and avoiding aggregation and precipitation

Active Publication Date: 2020-10-30
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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  • Preparation method of metreleptin
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  • Preparation method of metreleptin

Examples

Experimental program
Comparison scheme
Effect test

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 preparation method of metreleptin, which specifically comprises the following steps: expressing metriptostine genes through a prokaryotic expression system to obtain an inclusion body, dissolving the inclusion body, and carrying out hydrophobic chromatography renaturation to obtain metriptostine protein. Inclusion body dissolution and hydrophobic chromatography renaturation are both carried out in a mild mode, and an antioxidant and / or a metal chelating agent can also be added in the process so as to inhibit oxidation and degradation of target protein. The metreleptinprepared by the method is easy to realize industrial amplification, high in renaturation efficiency, high in yield and good in 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 Applications(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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