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Intracellular analysing and detecting method of peptidyl-propyl-cis/trans isomerase activity

A detection method and isomerase technology, which can be used in biochemical equipment and methods, microbial determination/inspection, biological testing, etc., and can solve problems such as intracellular analysis methods without PPIases activity

Inactive Publication Date: 2004-02-11
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the particularity of the reactions catalyzed by PPIases, there has been no report on the intracellular analysis of PPIases activity.

Method used

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Examples

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Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Escherichia coli cell expression system PPIases intracellular activity analysis and detection

[0031] Ribonuclease T1 (RNase T1) was selected as the intermediary protein for detecting the intracellular activity of PPIases. RNaseT1 contains four X-Pro bonds, two in the cis configuration and two in the trans configuration. The correct folding of this enzyme must be realized on the basis of the formation of the correct configuration of the X-Pro bond under the catalysis of PPIases.

[0032] Design a connecting peptide connecting RNase T1 and green fluorescent protein (GFP), the length is 6 amino acids, the sequence is: proline-proline-valine-alanine-threonine-methionine (pro, pro , val, ala, thr, met), the corresponding base sequence is: CCA CCG GTC GCC ACC ATG.

[0033] Use a nucleic acid synthesizer to synthesize primers with oligonucleotides corresponding to this linking peptide, and connect the nucleic acid sequence corresponding to the linking peptide to...

Embodiment 2

[0036] Observing the cultured Escherichia coli cells under a fluorescent microscope, obvious green fluorescence of GFP can be seen, indicating that there is PPIases activity in the Escherichia coli cells, and the RNase T1-GFP fusion protein is expressed and correctly folded. Example 2. Analysis and detection of intracellular activity of PPIases in Streptomyces expression system

[0037] According to the structural characteristics of the shuttle plasmid vector pAX 05a of Escherichia coli and Streptomyces, synthetic primers were designed.

[0038] Using the PCR method, the RNase T1-GFP fusion gene was obtained from the plasmid vector containing the RNase T1-GFP fusion gene constructed in Example 1 and a new restriction site was introduced.

[0039] Using the gene recombination method, the RNase T1-GFP fusion gene was inserted into pAX 05a to obtain the recombinant Escherichia coli and Streptomyces shuttle plasmid vector pAX 5aT1GFP, and transformed into Escherichia coli (E.coli ...

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Abstract

The present invention belongs to biotechnology, and the intracellular analysis and detection process of peptidyl-propyl-cis / trans isomerase activity includes constructing fusion gene through fusion of protein or polypeptide, which contains X-Pro bond and realizes correct folding via cis / trans isomerization under PPIases catalysis, with green fluorescent protein (GFP); expressing fusion protein; fluorescent observation of the green fluorescence of GFP; and indicating and analyzing the activity of PPIases inside microbe, plant and animal cell. The said process may be used in PPIases activity detection inside live cell, and the GFP fluorescence may be observed directly with fluorescent microscope to determine intracellular PPIases activity simply, flexibly and intuitively. The cell introduced into fusion gene may be used directly as the type culture for relevant PPIases research and the type culture may be proliferated and propagated via microbe and cell culturing process for long termuse.

Description

(1) Technical field [0001] The invention relates to a method for intracellular analysis and detection of peptidyl-prolyl-cis / trans isomerase activity, belonging to the field of biotechnology. (2) Background technology [0002] Peptidyl-prolyl cis / trans isomerases (PPIases for short) are ubiquitous enzymes involved in protein folding in cells from microorganisms to higher organisms. This enzyme can accelerate the cis-trans isomerization reaction of the amide bond (X-Pro) formed by any amino acid (X) in the peptide chain and proline (Pro) both inside and outside the cell. According to the homology of amino acid sequence and the ability to bind to specific immunosuppressant drugs, PPIases can be divided into three families with significantly different structures: cyclophilins (cyclophilins), FK506-binding proteins (FKBP's) and paulin ( Parvulins). [0003] PPIases are one of the hotspots in the field of protein folding research in recent years. Meanwhile, Cyclophilins and FK...

Claims

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Application Information

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IPC IPC(8): C12Q1/533C12Q1/68G01N33/52
Inventor 吉爱国J·W·恩格斯
Owner SHANDONG UNIV
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