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Self-fused concatenation protein modification method and application thereof

A modification method and protein modification technology, which can be used in the development of self-fusion tandem protein modification methods and the application field of protein modification, which can solve problems such as toxic effects and reducing protein biological activity.

Pending Publication Date: 2021-12-03
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, whether it is a very mature PEGylation technology or the fusion of HSA and Fc, it is necessary to introduce foreign macromolecules for modification, which may lead to immunogenicity, reduced protein biological activity and other potential toxic effects

Method used

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  • Self-fused concatenation protein modification method and application thereof
  • Self-fused concatenation protein modification method and application thereof
  • Self-fused concatenation protein modification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Biosynthesis and physicochemical property characteristics of embodiment 1 GFP multimer

[0030] Genes encoding GFP monomeric GFP 1, dimeric GFP 2 and trimeric GFP3 (containing 6×His tag) were synthesized by Sangon Biotechnology (Shanghai, China) and successfully cloned into the pET-25b(+) vector . The sequence of the GFP protein is shown in SEQ ID No:1. Multimeric GFP subunits are separated by flexible linkers GGGGS. After confirmation by gene sequencing, the plasmid was transformed into Escherichia coli BL21 (DE3) strain for expression. Before large-scale expression, the transformed monoclonal bacteria were first inoculated in 50 mL TB medium (containing 100 μg / mL ampicillin), and cultured overnight at 37° C. and 250 rpm with shaking. The next day, it was transferred into 1L of fresh TB medium (in a 2L shake flask, the concentration of ampicillin was 100 μg / mL) for large-scale culture and induction of expression. The specific steps are as follows: Firstly, shake cu...

Embodiment 2

[0036] The biological activity analysis of embodiment 2 GFP multimer

[0037] Protein fluorescence spectrum (480nm-570nm) was analyzed and measured by Varioskan Flash microplate reader (Thermo Scientific, USA), and the excitation wavelength was 460nm. The protein fluorescence value was measured at an excitation wavelength of 460nm and an emission wavelength of 507nm. At the same mass concentration (the protein test concentration is 0.25mg / mL), the retention rate of fluorescence intensity per unit of GFP is obtained by calculation, and the calculation formula is: fluorescence retention rate of multimer per unit of GFP = fluorescence value of multimer / GFP The fluorescence value of 1 x 100%, where the retention rate of GFP fluorescence intensity per unit in GFP 1 is 100%. At the same molar concentration (the protein test concentration is 10 μM), the relative amount of GFP contained in each unit concatemer is obtained by calculation, and the calculation formula is: the amount of ...

Embodiment 3

[0039] The in vitro thermal stability analysis of embodiment 3 GFP multimer

[0040] The in vitro thermal stability of the protein was tested by heat-denaturing the protein at 90°C for 2 minutes and refolding at room temperature. Before the test, adjust the fluorescence concentration of the sample to be the same (the final protein fluorescence value measured by a microplate reader is 2500, which is equivalent to the protein concentration of GFP 1, GFP 2 and GFP 3 10, 7 and 5 μM), and the test method is the same as the implementation Example 2. After heating at high temperature for 2 minutes, the protein fluorescence value was measured at given time (1, 5, 10, 15, 20, 25, 30, 45, 60 and 120 min).

[0041] Such as image 3 , GFP 3 recovers fluorescence faster than GFP 2, and GFP 2 recovers fluorescence faster than GFP1 ( image 3 a). After being placed at room temperature for 2 hours, the recovery fluorescence of GFP3 (53%) and GFP2 (22%) was 4.2 times and 1.8 times that of ...

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Abstract

The invention discloses a simple and universal protein modification method, in particular to a self-fused concatenation (SEC) protein modification method, which is used for improving the biological activity and the pharmaceutical property of protein. According to the invention, a group of GFP monomers, GFP dimers and GFP trimers are obtained through a genetic engineering technology. The GFP polymer can significantly improve the in-vitro biological activity and thermal stability of the GFP monomer and the residence time of mouse tumors, and the protein biological activity, thermal stability and tumor residence time are in positive correlation with the number of self-fused concatenation proteins. In addition, the IFN monomer, the IFN dimer and the IFN trimer are synthesized through the SEC technology, and the IFN polymer can remarkably improve the in-vitro biological activity, the in-vivo half-life period and the anti-tumor effect of the IFN monomer. The results show that the SEC can be used as a choice to replace the existing PEGylation or albumin fusion technology, and can be widely applied to other protein or small peptide drugs to improve the pharmacological characteristics, so that the long-acting protein or polypeptide drugs are successfully designed.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to the development of a self-fusion tandem protein modification method and its application in protein modification. Background technique [0002] Compared with small molecule drugs, proteins and peptides have high specificity and low toxicity, and have great potential for medical treatment in clinic. At present, a variety of FDA-approved therapeutic drugs (such as etanercept, insulin glargine, pefloxacin, bivalirudin, cyclosporine and octreotide, etc.) have been used in tumors, immunity, viral diseases and endocrine diseases. and other fields have been applied. However, proteins and peptides have problems such as poor stability, strong immunogenicity, and short half-life. [0003] Many strategies have been developed to partially address the above issues and improve the delivery efficiency of proteins / peptides. For example, covalently binding non-toxic polymer polyethylene g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/21A61K47/55A61P35/00
CPCC07K14/43595C07K14/555A61K38/21A61K47/55A61P35/00C07K2319/35
Inventor 胡瑾
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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