ApcE2 protein mutant and application thereof

A protein mutant and mutant technology, applied in the application, bacterial peptides, biochemical equipment and methods, etc., can solve the problems of affecting labeling, deletion modification, insufficient stability, etc., to achieve good solubility, improved solubility, and high unit yield. Effect

Active Publication Date: 2019-04-02
GUANGZHOU TEBSUN BIO TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005]Because ApcE2 is a membrane protein, it needs to be dissolved under the condition of high concentration of denaturant (3.5mol / L urea); it is not stable enough at room temperature, so it will affect the labeling
Existing literature (Miao Dan. Research and Molecular Evolution of Red-shifted Phycobiliproteins and Phytochromes [D]. 2017.) reported: truncation of the N-terminal and C-terminal of the protein and partial deletion of the loop (77-161) , did not increase protein solubility

Method used

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  • ApcE2 protein mutant and application thereof
  • ApcE2 protein mutant and application thereof
  • ApcE2 protein mutant and application thereof

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Experimental program
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Effect test

Embodiment 1

[0046] On the basis of the v0 sequence (amino acid sequence such as SEQ ID No. 1), the hydrophobic amino acid sites (R164K, S165A, L47E, L51N, L54T) outside the loop were mutated to obtain the v1-v4 mutant sequences, amino acid sequences respectively. As shown in SEQ ID No.2 to SEQ ID No.5.

[0047] On the basis of the mutant v4, the hydrophobic amino acid sites (L91T, V92S, L134E, L137D) in the loop were mutated to obtain the v5-v6 mutant sequences respectively, and the amino acid sequences are as shown in SEQ ID No.6~SEQ ID No. 7 is shown.

[0048] On the basis of mutant v6, the hydrophobic amino acid sites (W133Y, K135R) in the loop were mutated to obtain the v7-v8 mutant sequence. The amino acid sequence is shown in SEQ ID No. 8 to SEQ ID No. 9, wherein The v7 mutant was named BDFP3.

[0049] On the basis of mutant v7, the mutant loop structure sequence (Δ88-106, Δ88-114, Δ88-120, Δ88-130) was partially deleted to obtain the v9-v12 mutant sequence. The amino acid sequenc...

Embodiment 2

[0062] Preparation of Phytochrome PΦB and Its Application in Labeling Mammalian Cells

[0063] The gene sequence encoding the mutant was substituted for ApcE2(24-245), co-transformed into Escherichia coli with the heme oxidoreductase gene ho1 and the phytochrome synthase gene hy2, and recombinantly expressed to generate the pigment protein PΦB-mutant, which was extracted and purified. The phytochrome PΦB can be extracted from the chromoprotein PΦB-mutant under denaturing conditions. Pigment crystals can also be obtained by further rotary evaporation.

[0064] Specifically, the purified pigment protein was taken and its absorption characteristic peak was diluted to OD=2, proteinase K with a final concentration of 20 μg / mL was added, and the protein was digested in a water bath at 37° C. for 1 h. The degree of enzymatic hydrolysis was measured by an absorption spectrometer. When the enzymatic hydrolysis was relatively complete, proteinase K was removed to obtain a solution cont...

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Abstract

The present invention discloses a wild type ApcE2 protein mutant. The mutant is characterized by being obtained by mutation of at least one amino acid in an amino acid sequence shown in a SEQ ID No.1,and wherein the amino acid mutation is selected from at least one of mutation of a amino acid site outside a loop, mutation of an amino acid site inside a loop, and a loop structure sequence with partial deletion mutation. The ApcE2 protein mutant of the present invention has a higher solubility than that before the mutation, and the unit yield expressed in Escherichia coli is remarkably improved. The ApcE2 protein mutant of the invention can be used for fluorescence labeling tissue localization, preparation and extraction of phytochrome P[phi]B, preparation of other phycobiliproteins, such as phycoerythrin PEB and phycocyanobilin PCB, and in vitro recombination of fluorescent phycobiliprotein. The preparation method of the present invention realizes higher unit yield and better solubility of the pigment protein.

Description

technical field [0001] The present invention relates to ApcE2 protein mutants and applications thereof. Background technique [0002] The development of modern biology has increasingly relied on optical techniques, such as fluorescence imaging, optical detection, and light-induced manipulation. For imaging and positioning of deep tissue in vivo, it is best to use fluorescence in the 650nm-900nm band. Because the light in this band has less autofluorescence, less light scattering and less tissue damage. In recent years, two types of proteins that have been studied more as fluorescent probes are the green fluorescent protein family and the bacterial phytochrome protein (BphP). [0003] In order to better adapt to the environment and capture more energy from the environment, a few cyanobacteria have slowly evolved phycobilisomes that can utilize far-red light. In these phycobilisomes, there are usually one to three allophycocyanin subunits different from the traditional nucl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/31
CPCC07K14/195
Inventor 吴明卢艳华付卫雷陈彦蓉夏坤
Owner GUANGZHOU TEBSUN BIO TECH DEV
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