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Luciferase active fragment and application thereof

An active fragment, luciferase technology, applied in the field of biochemistry and cell biology research, can solve the problems of poor stability of enzyme protein, limited application range, less research and reports, etc.

Active Publication Date: 2011-06-01
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In comparison, these luciferases have relatively large molecular weights, and the stability of the enzyme protein in solution is poor, and some require co-activating factors such as ATP and Mg when used. 2+ ion or Ca 2+ ions, etc., which largely limits their range of application
[0004] Gaussia luciferase (Gaussia Luciferase, referred to as GLuc) is a new type of luciferase isolated from marine copepods, and there are few studies and reports on it at present

Method used

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  • Luciferase active fragment and application thereof
  • Luciferase active fragment and application thereof
  • Luciferase active fragment and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1. Primary sequence analysis of Gaussia luciferase (GLuc) and mass spectrometric identification of disulfide bonds

[0083] The known primary sequence of GLuc was analyzed, and the position of the disulfide bond was confirmed by mass spectrometry.

[0084] The test results showed that: the primary sequence of GLuc ( Figure 1A ) contains a total of 185 amino acid residues; its N-terminus is a 17Aa signal peptide sequence, which directs secreted expression; the core enzyme (which ranges from 44 to 185Aa in the sequence) has two highly homologous substructures Each domain contains five cysteine ​​residues; as determined by the Ellman method, GLuc does not contain free cysteine ​​sulfhydryl groups and forms five pairs of disulfide bonds. Further identification by mass spectrometry confirmed three pairs of disulfide bonds: C2-C3, C4-C5 and C9-C10, and the other two pairs of disulfide bonds have not yet been identified.

Embodiment 2

[0085] The artificial synthesis of embodiment 2.Gluc whole gene and Gluc 148DNA sequence, its cloning, expression and target ratio of purification

[0086] According to the codon preference of prokaryotes and eukaryotic mammals, optimize part of the codons of Gluc whole gene and Gluc 148 (Gluc 38-185) DNA sequence, so that it can be expressed efficiently in prokaryotic cells and mammalian cells . The codon-optimized DNA sequences of Gluc 185 and Gluc 148 are shown in Figure 1B In , the comparison of the DNA sequence of Gluc 148 with the original gene sequence is shown in Figure 1C middle.

[0087] The obtained optimized GLuc and GLuc148 genes were respectively cloned into pET-22b(+) plasmid (purchased from Novagen), and the plasmid was used to transform Escherichia coli Origami B strain (purchased from Novagen), and cultivated overnight at 37°C. The culture was inoculated into medium (LB medium), cultured at 37°C for 3 hours, added with IPTG (final concentration 50 μM...

Embodiment 3

[0089] Example 3. Fluorescence emission spectrum of active fragment GLuc 148

[0090] The fluorescence luminescence spectrum of Gluc 148 was studied using a fluorophotometer (available from Varian, model Cary Eclipse). The solution used for testing was 50mM PBS, pH7.8, 500mM NaCl, the concentration of GLuc148, in the reaction system, at room temperature Under the catalysis of coelenterazine (final concentration 1 μM) oxidative luminescence (reaction time 10 seconds). Fluorescence emission spectra were acquired between 400-580 nm.

[0091] Wild-type Gaussia luciferase is based on coelenterazine (Coelenterazine) (see structure Figure 3A ) as the substrate luciferase, catalyzing the oxidation reaction of the substrate (such as Figure 3B shown), the reaction produces CO 2 , and at the same time emit blue light with a wavelength of 480nm. Figure 3C For the luminescence spectrum of the purified active fragment GLuc 148 when it catalyzes the oxidation reaction of coelenteraz...

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Abstract

The invention obtains a Gaussia Luciferase (GLuc) active fragment by deleting 37 amino acids at the N-terminal of the GLuc, which is named as GLuc 148. Being the same as the wild type Gluc, the GLuc 148 has high luminescence activity and can achieve stable biochemical properties without the presence of complementary factors, but the GLuc 148 has a smaller molecular weight and performs non-secretory type expression in eukaryocytes. Thus, the GLuc 148 is more suitable for being used as a report factor and a fluorescent energy donor, and can be used in the technical fields of cell and in-vivo imaging, high-flux screening, microorganism detecting and the like.

Description

technical field [0001] The invention relates to the fields of biochemistry and cell biology research, in particular to the transformation and application of Gaussia luciferase activity core fragment GLuc 148. Background technique [0002] Luciferase is a general term for a class of enzymes that catalyze the oxidation and luminescence of luciferin or aliphatic aldehydes in organisms. Due to its unique luminescent characteristics and easy, sensitive and rapid detection, the luciferase gene has become a widely used reporter gene (reporter) in genetic engineering; And the application of environmental monitoring, microbiological detection, etc. has also attracted more and more attention. [0003] At present, the most studied and commonly used luciferases are mainly: Bacterial Luciferase (BLuc for short), Firefly Luciferase (Firefly Luciferase, FLuc) and Renilla Luciferase (Renilla Luciferase, RLuc). In comparison, these luciferases have relatively large molecular weights, and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12Q1/68C12Q1/66C12Q1/04G01N33/68G01N21/64
Inventor 胡红雨李海音郑学明
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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