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Flavin mononucleotide based fluorescence protein having modified performances

A technology of flavin mononucleotide and protein, applied in the field of flavin mononucleotide combined with fluorescent protein, to achieve the effect of improving thermal stability

Active Publication Date: 2015-05-13
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, many computational methods have been used to test model systems, and there are few reports on the use of these computational methods to improve protein stability.

Method used

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  • Flavin mononucleotide based fluorescence protein having modified performances
  • Flavin mononucleotide based fluorescence protein having modified performances
  • Flavin mononucleotide based fluorescence protein having modified performances

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Selection of rationally designed mutants:

[0022] The present invention adopts foldX (Guerois, R.; Nielsen, J.E.; Serrano, L.J.Mol.Biol.2002) by starting from the LOV subunit crystal structure (pdb: 2PR5) of Bacillus subtilis flavin mononucleotide combined with blue fluorescent protein YtVA , 320, 369.) software for stable single mutant screening. The steps are briefly described as follows: First, use the 'RepairPDB' command in foldX to minimize the energy of the subunit structure, and then use the 'PositionScan' command to perform 19 kinds of natural amino acid mutations except wild protein amino acids at each position of the optimized structure, And to calculate the impact of mutations on thermal stability, 712 mutations of 94 sites in the above-identified sites showed improved thermal stability. Then, based on the fact that there are multiple mutations at a certain site, several relatively stable sites are selected; at the same time, the mutated amino acid involves...

Embodiment 2

[0070] According to the single mutations obtained above, double mutation combinations were performed, including N107F_N124F, N107Y_N124Y, and N107Y_V120I. These combined mutations were again subjected to the fluorescence detection and Tm fitting process of the above steps, and then the influence of the combined mutants on the thermal stability Tm value was obtained as shown in the table 2.

[0071] Among them, N107F_N124F and N107Y_N124Y showed higher stability than any single mutation in the double mutation, while the Tm value of N107Y_V120I was comparable to that of N107Y, indicating that this combination had no additive effect. Then, based on the N107Y_N124Y double mutation, further modifications including triple mutations N107Y_N124Y_H22W and N107Y_N124Y_M111F were performed. Among them, the N107Y_N124Y_H22W mutation is slightly less stable than the double mutation N107Y_N124Y, while the triple mutation N107Y_N124Y_M111F has enhanced thermal stability compared to the doubl...

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Abstract

The invention related to a modified fluorescence protein, and particularly relates to a flavin mononucleotide based fluorescence protein FbFP having modified performances at high temperatures. In concrete, the fluorescence protein is a flavin mononucleotide based blue fluorescence protein YtVA LOV subunit which has one or more amino acid site mutations. According to the fluorescence protein provided by the invention, a large-scale computer screening method and an experiment verification method are combined for the first time to design a thermally-stabilized mutation for the fluorescence protein FbFP, stable mutants can be used for studying fluorescence labeling for a high-temperature microorganism system, particularly an anaerobic system.

Description

technical field [0001] The invention relates to a modified fluorescent protein, specifically a flavin mononucleotide-binding fluorescent protein FbFP with improved performance at high temperature. Background technique [0002] Green fluorescent protein and its derivatives need oxygen to synthesize their chromophores (Tsien, R.Y. Annu Rev Biochem 1998, 67, 509.), thus limiting their application in anaerobic systems. The discovery of the blue fluorescent protein YtVA based on flavin mononucleotide binding from Bacillus subtilis provides a new way to solve this problem (Losi, A.; Polverini, E.; Quest, B.; Gartner, W. Biophys.J.2002,82,2627.), the protein can emit fluorescence in both anaerobic and aerobic microorganisms (Drepper, T.; Eggert, T.; Circolone, F.; Heck, A.; Krauss, U .; Guterl, J.K.; Wendorff, M.; Losi, A.; Gartner, W.; Jaeger, K.E. Nat Biotechnol 2007, 25, 443.). YtvA consists of 261 amino acids, mainly including a LOV (light, oxygen, voltage) subunit (residues ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/32C07K14/21
Inventor 姚礼山宋乡飞王业飞
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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