Multiple improved orange/red fluorescence protein

A red fluorescent protein and protein technology, which is applied in the field of improved orange/red fluorescent protein, can solve the problems of not being able to meet the requirements of confocal imaging, shortening of photostability, etc.

Active Publication Date: 2016-08-03
北京义翘神州科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after DsRed is mutated into a monomer, its photostability is greatly shortened, which cannot meet the requirements of confocal imaging, and because the structure of the luminescent body is changed, it is easy to form more red fluorescent proteins with different colors.

Method used

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  • Multiple improved orange/red fluorescence protein
  • Multiple improved orange/red fluorescence protein
  • Multiple improved orange/red fluorescence protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The construction of embodiment 1.DsRed mutant library:

[0029] Through the analysis of the protein structure of DsRed (see SEQ.ID.NO: 1 for the amino acid sequence) by Discoverystudio4.0 software, find out 48 sites that may affect the structure of DsRed, these 48 sites are: R2, S4, K5, E10 , V16, R17, T21, E32, R36, H41, N42, V44, K47, Q64, F65, Q66, V71, V73, K83, L85, F91, E99, C117, F118, F124, I125, V127, T147, L150 , R153, V156, E160, I161, H162, K163, A164, L174, V175, F177, S179, I180, Y192, Y194, S197, I210, T217, G219, L225, stagger these 48 points into 12 groups (Table 2), performing saturation mutations on the amino acids at each site in each group, so that 12 groups of saturation mutation gene pools were obtained; in addition, the DsRed gene was randomly mutated by the error-prone PCR method to obtain a group of mutation gene pools; 13 groups of mutated genes were randomly recombined by DNAShuffling method, inserted into pQE30 vector, and pQE30-DsRed-DS li...

Embodiment 2

[0035] Screening of embodiment 2.DsRed mutant clones:

[0036] Spread the DsRed-DS library evenly on the ZYM-AT self-inducing medium plate (1% tryptone, 0.5% yeast extract, 25mM Na 2 HPO 4 , 25mMKH 2 PO 4 , 50mM NH 4 Cl, 5mM Na 2 SO 4 , 0.5% glycerol, 0.05% glucose, 0.2% a-lactose, 2mM MgSO 4 , 0.2x trace elements, 1% agar and 50 μg / mL Amp), cultured overnight at 30°C, the single clones in the DsRed-DS library expressed mutant fluorescent proteins, showing different brightness and colors. Observe the plate and circle the brighter red colonies with a marker. The next step is to prepare ZYM-AT self-induction liquid medium, and add 200 μl ZYM-AT liquid medium to each well of a 96-well deep-well plate. Then these bright red colonies were picked out and inoculated into 96-well plates, a total of 1820 clones were picked, and all deep-well plates were placed in a shaker at 30°C and 200rpm for 16 hours for fluorescent protein expression. The overnight expression bacterial sol...

Embodiment 3

[0040] Construction and expression of embodiment 3.sRFP mutant fluorescent protein and mOrange2 fluorescent protein eukaryotic expression vector:

[0041]Use the primers sRFP-inf-F: 5'GGTACCGCTAGCGGATCCATGGATAGCACTGAGAACGTCA3' (SEQ.ID.NO: 10) and sRFP-inf-R: 5'GGCCGCCTCTAGACTCGAGCTACTGGAACAGGTGGTGGC3' (SEQ.ID.NO: 11) to use these three mutants as templates respectively The sRFP2, sRFP10 and sRFP12 genes were amplified and inserted into the eukaryotic expression vector pCMV3 by infusion enzymes, and the correct plasmids pCMV3-sRFP2, pCMV3-sRFP10 and pCMV3-sRFP12 were identified. Purify the pCMV3-sRFP2, pCMV3-sRFP10 and pCMV3-sRFP12 plasmids with transfection grade purity to transfect 293H cells. The specific steps are: 293H cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% carbon dioxide. Before transfection, cells were cultured at 1×10 5 The density of cells / well was seeded in a 24-well plate, and used for transfection after 24 hours of cultu...

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Abstract

The invention relates to multiple improved orange / red fluorescence protein. By modification of amino acid sequence of red fluorescence protein DsRed from Discosoma sp., the obtained novel fluorescence protein has different characteristics from a wild type. A brighter monomer structure orange fluorescence protein similar to mOrange2 is obtained, and the fluorescent brightness is 2.0 times higher than fluorescent brightness of the mOrange2. According to the invention, a novel purple red fluorescence protein is also obtained, and the fluorescence protein has specific maximum excitation wavelength and maximum emission wavelength. The invention also relates to research of the improved fluorescence protein applied in multi-fields. Positioning, tracing, functional display and the like of target proteins in cells, subcells and in-vivo tissues can be analyzed.

Description

technical field [0001] The present invention relates to some improved orange / red fluorescent proteins. By modifying the DsRed sequence from the coral polyp Discosomasp. red fluorescent protein, the resulting novel fluorescent protein has significantly different colors (excitation spectrum and emission spectrum), fluorescence intensity, etc. from the wild type. features. The present invention also relates to the cloning and expression of the above-mentioned fluorescent protein gene and its ability to be fused with the protein at the N-terminal or at the C-terminal, expressed in cell lines or living animals, and capable of positioning the target protein in cells, sub-cells and living tissues , Tracing, function display, etc. for a variety of analysis. Background technique [0002] Fluorescent proteins serve as molecular tags and have broad applications in analytical biotechnology and intracellular molecular tracking. Especially in the application of cellular molecular imagin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/85C12N15/867
Inventor 孙春昀谢良志饶木鼎赵淑环徐明明陈军
Owner 北京义翘神州科技股份有限公司
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