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Thermal stable protein (polypeptide) and preparation method thereof

A thermal stability and protein technology, applied in the biological field, can solve the problems of inapplicable improvement of the stability of soluble biological macromolecules, inability to improve the stability of soluble macromolecules, and inability of amorphous calcium phosphate to exist stably. The method is simple and the effect of improving stability

Inactive Publication Date: 2017-10-27
CHONGQING MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Previously, an applicant applied for the related technology of "a genetically engineered heat-stable vaccine and its preparation method", the application number is 201310278495.X, and the application date is 2013.07.02. This technology is mainly used in the production of virus particle vaccines. However, it is not suitable for the improvement of the stability of soluble biomacromolecules, because the virus itself is a particle molecule, so it can form a mineralized shell by expressing mineralized peptides on the surface, while soluble molecules cannot be directly mineralized on the surface, so this technology cannot improve The stability of soluble macromolecules, and the amorphous calcium phosphate formed by this technology cannot exist stably in liquid solution, and cannot make proteins (polypeptides) thermally stable

Method used

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  • Thermal stable protein (polypeptide) and preparation method thereof
  • Thermal stable protein (polypeptide) and preparation method thereof
  • Thermal stable protein (polypeptide) and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, the preparation of the protein with self-mineralization ability

[0048] In this example, the pneumolysin ΔPly protein with amino acid 146 removed was used as the model skeleton, and the pET28a plasmid was used as the carrier to construct pET28a(+)-ΔPly-PA44, pET28a(+)-ΔPly-NW, pET28a(+) -ΔPly–N6, pET28a(+)-ΔPly–W6 recombinant expression plasmids, and then the recombinant expression plasmids were transformed into E. coli, and expressed, identified and purified in E. coli

[0049] (1) Mineralization peptide linked to ΔPly protein

[0050] 1. Construction of recombinant expression plasmids

[0051] (1) Material:

[0052] Plasmid pET28a(+) was purchased from Novagen, Prime Star high-fidelity enzyme, dNTPs, Buffer, MgCl for PCR 2 Purchased from Dalian Bao Biological Technology Co., Ltd., PTC-200 PCR instrument is a product of Perkin Elmer, and RG-3000 is a product of CorbettResearch.

[0053] (2) Design and synthesis of primers:

[0054] Using Streptococc...

Embodiment 2

[0111] Example 2. Calcium phosphate self-mineralization and biometric analysis of recombinant protein ΔPly-PA44

[0112] (1) Mineralization of recombinant protein

[0113] Add CaCl to the ΔPly-PA44 protein solution (adjust the protein concentration to 100ng / ml) 2 , so that Ca 2+ When the ion concentration reaches 5 μmol / ml, add Mgcl 2 ions, making Mg 2+ The ion concentration is 5μmol / ml; finally add (Na) 2 HPO 4 / NaH 2 PO 4 Make PO 4 3- The ion concentration was 3 μmol / ml, and the system was rotated and stirred with magnetic beads at 4°C and pH=6.5 for 2 hours to prepare a thermally stable mineralized protein.

[0114] In the above preparation process, the protein, Ca 2+ Ions, Mg 2+ ions and PO 4 3- The ratio of ions is 100(ng):5(umol):5(umol):3(umol).

[0115] (2) Biometric analysis

[0116] (1) Centrifuge the mineralized protein ΔPly-PA44 (5000g; 5min) to obtain the precipitate and supernatant, resuspend the precipitate with 40ul PBS, add 10ul 5×loading buffer...

Embodiment 3

[0124] Example 3: Self-mineralization and biometric analysis of recombinant protein ΔPly-NW calcium phosphate

[0125] (1) Mineralization of recombinant protein

[0126] Add CaCl to the ΔPly-PA44 protein solution (adjust the protein concentration to 200ng / ml) 2 , so that Ca 2+ When the ion concentration reaches 7μmol / ml, add Mgcl 2 ions, making Mg 2+ The ion concentration is 7μmol / ml; finally add (Na) 2 HPO 4 / NaH 2 PO 4 Make PO 4 3- The ion concentration was 5 μmol / ml, and the system was rotated and stirred with magnetic beads at 4°C and pH=6.5 for 2 hours to prepare a thermally stable mineralized protein.

[0127] In the above preparation process, protein, Ca 2+ Ions, Mg 2+ ions and PO 4 3- The ratio of ions is 200(ng):7(umol):7(umol):5(umol).

[0128](1) Biometric analysis

[0129] (1) Centrifuge the mineralized protein ΔPly-NW (5000g; 5min) to obtain the precipitate and supernatant, and resuspend the precipitate with 40ulPBS,

[0130] Add 10ul 5×loadingbuff...

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Abstract

The invention discloses thermal stable protein (polypeptide) and a preparation method thereof, and particularly relates to protein (polypeptide) with self-mineralizing capacity. The protein (polypeptide) contains a polypeptide which is capable of inducing organic matters to mineralize and is introduced by adopting the gene engineering technology. The thermal stable protein (polypeptide) is characterized in that nano particles having certain sizes can be formed by the protein (polypeptide) through biomimetic mineralization, and an amorphous calcium phosphate layer is formed outside the protei (polypeptide) and can be used for coating the protein (polypeptide) molecules, so that the thermal stability of the protein (polypeptide) can be improved, and the degradation effect of protease can be resisted.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a thermostable protein (polypeptide) and a preparation method thereof. Background technique [0002] At present, protein (polypeptide) drugs are widely used in clinic because of their advantages of high activity, high specificity, low toxicity and clear biological function. Protein (polypeptide) drugs can be divided into cytokines, enzymes, antibodies, vaccines, etc. With the development of protein (polypeptide) omics and molecular biology, the development and application of protein (polypeptide) drugs used in the treatment of various diseases has become a hot spot in the biomedical industry. [0003] Compared with small molecular compound drugs, protein (polypeptide) drug molecules are large, usually thousands or tens of thousands or even hundreds of thousands of daltons. The function of protein (polypeptide) is composed of various functional groups, but these functional groups ar...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70
CPCC07K14/3156C07K2319/00C12N15/62C12N15/70
Inventor 尹一兵张雪梅吴静雯吴凯峰肖云菊
Owner CHONGQING MEDICAL UNIVERSITY
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