Endothelium chalone mutant containing non-natural amino acid and derivatives thereof

A technology of unnatural amino acids and endostatin, applied in the biological field, can solve the problems of few modified products, inability to modify at fixed points, difficult modification conditions, etc., and achieve the effect of simplifying the steps of refolding and purification

Inactive Publication Date: 2008-09-17
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the modifications for endostatin are amino modification and N-terminal modification. These modification methods either cannot be modified at a specific point, resulting in inhomogeneous product properties, or the modification conditions are difficult to control, and the modified products are less and unstable.

Method used

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  • Endothelium chalone mutant containing non-natural amino acid and derivatives thereof
  • Endothelium chalone mutant containing non-natural amino acid and derivatives thereof
  • Endothelium chalone mutant containing non-natural amino acid and derivatives thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Site-directed introduction of p-acetylphenylalanine in endostatin

[0020] The unnatural amino acid site-directed introduction system includes a suppressor tRNA (tRNA Tyr ) and aminoacyl tRNA synthetase (Mj TyrRs), which are encoded and expressed by the stringent plasmid pAC-tRNA and the relaxed plasmid pBR-TyrRS, respectively. The endostatin gene underwent the following three changes: ①The inactive site Phe32 was mutated into the amber codon TAG; ②The expression was initiated by T7 promoter and T7 terminator; ③The His6 tag was added to the N-terminus of endostatin. The endostatin gene with the above three changes was constructed on the vector pAC-tRNA, named pAC-tRNA-ES. Competent cells DH10B were co-transfected with pAC-tRNA-ES and pBR-TyrRS, coated with two antibiotics, Tet (10 μg / mL) and Amp (100 μg / mL), and incubated overnight at 37°C for 12 hours. Pick a single colony from the double-transformation plate and inoculate it in LB medium with two antibiotics, shake ...

Embodiment 2

[0022] Site-directed introduction of p-azidophenylalanine in endostatin

[0023] The unnatural amino acid site-directed introduction system includes a suppressor tRNA (tRNA Tyr ) and aminoacyl tRNA synthetase (Mj TyrRs), which are encoded and expressed by the stringent plasmid pAC-tRNA and the relaxed plasmid pBR-TyrRS, respectively. The endostatin gene underwent the following three changes: ①The inactive site Phe32 was mutated into the amber codon TAG; ②The expression was initiated by T7 promoter and T7 terminator; ③The His6 tag was added to the N-terminus of endostatin. The endostatin gene with the above three changes was constructed on the vector pAC-tRNA, named pAC-tRNA-ES. Competent cells DH10B were co-transfected with pAC-tRNA-ES and pBR-TyrRS, coated with two antibiotics, Tet (10 μg / mL) and Amp (100 μg / mL), and incubated overnight at 37°C for 12 hours. Pick a single colony from the double-transformation plate and inoculate it in LB medium with two antibiotics, shake t...

Embodiment 3

[0025] Isolation and purification of endostatin mutant with unnatural amino acid and its detection by Western-blotting

[0026]The above ultrasonic supernatant was separated and purified by Ni-NTA column. First equilibrate 10 column volumes with the above-mentioned ultrasonic buffer, then load the above-mentioned ultrasonic supernatant, and use 50mM imidazole, 100mM imidazole, 200mM imidazole, 300mM imidazole pH8.0, and 0.1M sodium phosphate eluent to elute respectively , and the 280nm light absorption of the eluate was detected by spectrophotometry, and a high absorption peak appeared at the concentration of 200mM imidazole. SDS-PAGE and Western-blotting analysis respectively

[0027] SDS-PAGE analysis: attached figure 1 For the electrophoresis diagram of the whole bacteria after induction, according to the attached figure 1 It was found that when no unnatural amino acid was added inside the medium, there was no expression of endostatin. Therefore, it was determined that ...

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Abstract

The invention relates to a non-natural amino acid-containing endostatin mutant and derivatives thereof, in particular to a non-natural amino acid-containing soluble endostatin expressed in prokaryotic expression system while the activity thereof is ensured. The endostatin mutant is endowed with new chemical property, and can specifically form the covalent linkage with other molecules through side chain of non-natural amino acid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and mainly relates to an endostatin mutant containing unnatural amino acids and its derivatives. The unnatural amino acids contained in the endostatin mutant can form specific covalent bonds with other molecules. connect. Background technique [0002] Angiogenesis is a multi-step process including proliferation, migration and differentiation of endothelial cells, degradation of extracellular matrix, formation of microvessels and sprouting of new capillaries. It is indispensable for embryonic development, organ formation, tissue regeneration, and wound repair. At the same time, angiogenesis is a necessary condition for tumor growth and metastasis. Endostatin is a kind of angiogenesis inhibitor capable of inhibiting the proliferation of endothelial cells, with a molecular weight of 20KD, and can specifically inhibit the proliferation and migration of endothelial cells. Studies have found that endost...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47A61K38/17A61P35/00
Inventor 姚文兵高向东陈明杰刘静娴田浤
Owner CHINA PHARM UNIV
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