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Detection method based on protein substrate and application thereof

A protein substrate and detection method technology, applied in the field of biochemistry, can solve the problems of inability to achieve high throughput, time-consuming, labor-intensive, termination, etc.

Inactive Publication Date: 2015-07-08
SHANGHAI SHENGFU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the difficulty in handling natural protein substrates, natural protein substrates are rarely used in protease-related biological assays.
One of the disadvantages of conventional natural protein substrates for protease activity assays is that the reaction usually needs to be terminated and the products then separated by electrophoresis or chromatography
These steps are laborious and time consuming, do not allow for high throughput and lack quantitative accuracy

Method used

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  • Detection method based on protein substrate and application thereof
  • Detection method based on protein substrate and application thereof
  • Detection method based on protein substrate and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Renin is a protease that cleaves Angiotensinogen to produce Angiotensinogen I polypeptide. The Renin-Angiotensinogen system plays an important role in regulating blood pressure. The amino acid sequence of Angiotensinogen was obtained from UniProtKB. To generate an N-terminal fluorescently labeled Angiotensinogen, a cysteine ​​was added to the N-terminal of Agiotensinogen, as in Figure 5 A shown. The amino acid sequence was optimized and synthesized by conventional methods. The gene was cloned into pE-SUMO vector through BsaI and XbaI, and transformed into Escherichia coli BL21 (DE3). This protein is expressed in fusion with SUMO protein, and the fusion protein has a 6×His tag at the N-terminus. The bacteria were cultured. When the OD600 reached 1.0, 0.5 mM IPTG was added to induce expression. The bacteria were resuspended with 25 mM Tris-HCl, 150 mM NaCl, pH 8.0, and disrupted by ultrasonic waves. Subsequently, the expressed protein was purified by Ni affinity chromat...

Embodiment 2

[0097] Beta-secretase (BACE) is one of two proteases that cleaves amyloid precursor protein to produce amyloid polypeptide 1-42. The human APP protein contains 770 amino acids, and the full-length APP contains several domains. The cleavage site of BACE is located between amino acids 671 and 672. The amino acid sequence of APP was obtained from UniProtKB. In this example, the amino acid sequence ranged from 365 to 699 aa. After synthesizing the gene sequence by conventional methods, the gene was cloned into the pTXB1 vector with NdeI and SpeI, and the target protein was directly combined with the contents of the vector. Peptide tag (intein) fusion. The constructed expression vector was transformed into Escherichia coli BL21 (DE3), and the bacteria were cultured. When the OD600 reached 0.6, 0.5 mM IPTG was added to induce expression. The bacteria were resuspended with 25 mM Tris-HCl, 150 mM NaCl, and pH 8.0. Ultrasonic fragmentation was performed, and then the expressed protei...

Embodiment 3

[0100] In this example, 5-TAMRA was labeled at the C-terminus of a shorter form of APP containing one domain (APP550-685) using the same method used to produce APP365-699-(5-TAMRA), as in Figure 12 shown. PCR was used to amplify the gene according to the conventional method, the gene was cloned into pTXB1 vector with NdeI and SpeI, and the target protein was directly fused with the intein tag (intein) carried by the vector. The constructed expression vector was transformed into Escherichia coli BL21 (DE3), and the bacteria were cultured. When the OD600 reached 0.6, 0.5 mM IPTG was added to induce expression. The bacteria were resuspended with 25 mM Tris-HCl, 150 mM NaCl, and pH 8.0. Ultrasonic fragmentation was performed, and then the expressed protein was purified by chitin affinity chromatography. At this time, the purified protein was retained on the chitin column. The resin was rapidly washed with 50mM MESNA in 25mM Tris-HCl, 150mM NaCl, pH 8.0, and then The column was p...

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Abstract

The invention discloses a detection method based on a protein substrate and application thereof. The detection method includes following steps: (1) preparing the protein substrate having a recognition site of a to-be-detected protease; (2) linking a detection component onto the recognition site of the to-be-detected protease of the protein substrate to form a site-specificity-marked protein substrate; (3) performing interaction to the site-specificity-marked protein substrate with the to-be-detected protease; and (4) detecting the interaction of the site-specificity-marked protein substrate with the to-be-detected protease according to the change of a signal of a specificity mark. The method can be used for screening compounds to achieve high throughput screening of drugs, thereby increasing development of the drugs.

Description

technical field [0001] The invention relates to a determination method in the field of biochemistry and its application, in particular to a protein substrate-based detection method and its application. Background technique [0002] Although the substrates of proteases in vivo are proteins with full-length amino acid sequences, most protease assays used in biological research and drug development employ polypeptides as substrates for proteases. A polypeptide is a small fragment of a protein (the sequence of which is derived from the protein). Although a polypeptide has a similar or even identical amino acid sequence to the corresponding fragment of its source protein, it still lacks many key structures and functions because it lacks the tertiary structure of a protein. Therefore, the use of peptides as substrates in drug screening, optimization and structure-activity relationship analysis of a target protease has many serious drawbacks. [0003] Polypeptide substrates are d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/37
Inventor 汪弋张成全
Owner SHANGHAI SHENGFU BIOTECH CO LTD
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