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Fused polypeptide mutant containing protein transduction domain and preparation method thereof

A technology for protein transduction domains and mutants, applied in the field of fusion polypeptide mutants containing protein transduction domains and its preparation, can solve problems such as loss of function, unfavorable drug production, and unstable protein expression

Inactive Publication Date: 2013-01-09
黄晨
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Problems solved by technology

Although the synthesized SARA fusion protein has certain functional activity, the expression of the protein is unstable and exists in the form of inclusion bodies, and the precipitated protein can only be used after renaturation, but renaturation often changes the properties of the protein and loses its function, which is not conducive to future of drug production

Method used

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  • Fused polypeptide mutant containing protein transduction domain and preparation method thereof
  • Fused polypeptide mutant containing protein transduction domain and preparation method thereof
  • Fused polypeptide mutant containing protein transduction domain and preparation method thereof

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Embodiment Construction

[0017] 1. In order to express the TAT-SARA / SBD fusion protein mutant of the present invention in the S1 plasmid, add His tag and SUMO tag in front of the fusion protein mutant. A nucleic acid fragment encoding TAT-SARA / SBD was designed and synthesized according to the customary codons of Escherichia coli:

[0018] CATATGATGC ATCACCACCA CCATCACTAT GCCCGTGCGG CGGCGCGTCA

[0019] GGCCCGTGCT TCTGGCGGCG GTTCAATGTC GGCGAGTAGT CAGAGTCCGA

[0020] ACCCGAACAA TCCGGCAGAA TATTGCTCCA CCATTCCGCC GCTGCAGCAA

[0021] GCGCAGGCCA GCGGCGCGCT GAGCTCTCCG CCGCCGACCG TGATGGTTCC

[0022] GGTCGGCGTT CTGAAACACC CGGGCACCGA AGTTCCGCAA CCGCGTTAAT

[0023] GAAAGCTT (SEQ ID NO: 5) (Figure 1).

[0024] 2. Plasmids S1 and M2 were obtained from Nanjing GenScript Biotechnology Co., Ltd., whose cloning sites are Nde I and Hind III, and the above-mentioned synthesized fragments were subcloned into plasmid S1. (figure 1)

[0025] 3. Induced expression of TAT-SARA / SBD fusion protein mutants

[0026] Transfo...

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Abstract

The invention provides a structural sequence and a construction method of a fused polypeptide mutant containing a protein transduction domain TAT (Transactivator of Transcription), a Smad anchor and a receptor-activated protein SARA peptide aptamer (SARA / SBD). The fused protein mutant contains 73 amino acids. The protein is subjected to fusion expression and purification, and purified TAT-SARA / SBD is taken as a tested medicament for performing an animal single-side ureteral ligation obstruction model experiment. As proved by a result, the tested medicament has a fibrosis reversing effect. The fused protein mutant can be subjected to efficient supernatant expression, is easy for purifying, is convenient for roducing, and is expected to be applied to treatment of kidney fibrosis.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, relates to a fusion polypeptide mutant containing protein transduction domain TAT, Smad anchor and receptor activation protein SARA peptide aptamer (SARA / SBD), and also relates to a cDNA sequence encoding the fusion polypeptide mutant and derivatives thereof, recombinant expression vectors comprising such cDNA sequences, host cells transformed with such recombinant expression vectors, and methods for preparing such recombinant proteins and their derivatives. The invention also relates to the construction method and application of the fusion protein mutant. Background technique [0002] Renal fibrosis is the ultimate common outcome of chronic kidney disease caused by different etiologies (including inflammation, injury, drugs, diabetes and genetic factors, aging, etc.). Various progressive kidney diseases, regardless of the primary disease, will eventually lead to renal fibrosis, progress to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K38/16A61K47/48A61K48/00A61P13/12A61K47/65
Inventor 黄晨孙世仁王汉民张鹏杜锐许国双刘晓渭刘宏宝何丽洁李嵘
Owner 黄晨
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