Biological orthogonal Raman in-situ detection method for conformation changes of protein

A bio-orthogonal and conformational change technology, which is applied in the detection of protein chemistry and conformational changes, can solve problems such as Raman spectral peak overlap, and achieve the effects of low interference of orthogonal Raman signals, simple data processing, and high sensitivity

Inactive Publication Date: 2017-07-07
PEKING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, in order to solve the problem of overlapping Raman spectrum peaks of biomolecules, the inventors of the applicant have studied the strategy of orthogonal Raman labeling, that is,

Method used

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  • Biological orthogonal Raman in-situ detection method for conformation changes of protein
  • Biological orthogonal Raman in-situ detection method for conformation changes of protein
  • Biological orthogonal Raman in-situ detection method for conformation changes of protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Detection of conformational changes of alkyne-containing HdeA protein in solution at different pH

[0043] HdeA is a molecular chaperone protein in the bacterial membrane stroma. In the physiological environment of pH 7, HdeA exists in the form of homodimerization; while in the physiological environment of pH 2, HdeA depolymerizes to expose its active site and bind to its substrate protein. In the physiological environment of pH 2, HdeA is in a disordered state, and its structure and conformational changes cannot be studied by X-ray crystallography. Penk is a lysine derivative containing an alkyne group (as shown in the figure below), and the orthogonal Penk-MbPylRS / tRNA can be used to insert Penk into the TAG site of the protein.

[0044]

[0045] Synthesis of AuNPs: Gold nanoparticles (AuNPs) sol were prepared by sodium citrate reduction method, in which HAuCl 4 The final concentration of sodium citrate was 0.01% (w / w). In boiling ultrapure water, add ...

Embodiment 2

[0053] Example 2 Detection of conformational changes of cell surface alkyne-containing EGFR protein before and after EGF stimulation

[0054] EGFR is an epidermal growth factor receptor protein located on the surface of cell membranes. The EGFR signaling pathway plays an important role in physiological processes such as cell growth, proliferation and differentiation, and is closely related to a variety of cancers. EGFR will dimerize after combining with epidermal growth factor EGF, and EGF mainly exists in the form of monomer after being released.

[0055] Synthesis of AuNPs: Prepared by sodium citrate reduction method.

[0056] Expression of alkyne-containing EGFR in cells: the codons at positions 210, 250, 356 or 440 of the EGFR protein were mutated to TAG. In the presence of the unnatural amino acid Penk, will contain engineered EGFR and Penk-MbPylRS / tRNA Pyl CUA The plasmid of the gene sequence was transiently transformed into HEK293T cells. After 40 hours, the cells ...

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Abstract

The invention relates to a biological orthogonal Raman in-situ detection method for conformation changes of protein, in particular to a method for detecting conformation changes of protein through a biological orthogonal Raman report group specificity marking. The method comprises the following steps that 1, unnatural amino acid with a biological orthogonal Raman report group is prepared; 2, the unnatural amino acid prepared in the step 1 is inserted into target protein through a gene extension technique to achieve biological orthogonal Raman report group marking on the target protein; 3, the protein obtained in the step 2 is detected through a biological orthogonal Raman technique. According to the method, the conformation changes of the protein with the biological orthogonal group under different environment conditions or activity conditions are sensitively and efficiently detected through a gene codon amplification technique and an SERS enhancement method, and a connection between an analysis solution and the protein conformation and function in a cell system is achieved.

Description

technical field [0001] The present invention relates to the detection of protein chemical and conformational changes, in particular to a method of using bio-orthogonal Raman reporter group to specifically label and detect proteins, using bio-orthogonal Raman technology to detect protein conformation changes in vitro, and using bio-orthogonal Raman Mann technology is a method for in situ detection of protein conformational changes in living cells. Background technique [0002] The spatial conformation of a protein molecule is the basis of its function and activity. The response of proteins to the environment and stimuli is through the conformational changes of proteins or protein complexes to realize the regulation of their functions and activities. The positions, orientations, and movements of some important amino acids undergo significant changes during the protein's allosteric process. Therefore, rapid, sensitive and efficient detection of protein conformational changes ...

Claims

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Application Information

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IPC IPC(8): G01N21/65
CPCG01N21/658
Inventor 陈兴李娅娅洪森炼林亮肖明杜逸飞
Owner PEKING UNIV
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