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Fusion protein of pellicle growing gene and green fluorescence albumen

A technology of epidermal growth factor and green fluorescent protein, applied in the fields of molecular biology and biomedicine, can solve problems such as difficult to develop proteins, affect the inherent folding form of fused proteins, and change protein activity

Inactive Publication Date: 2008-01-02
SHANGHAI INST OF ONCOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] While fusing green fluorescent protein (GFP) or other fluorescent proteins to a protein of interest is a well-established technique, it is also understood that such fusion techniques can affect the intrinsic folded form of the fused protein
GFP can not only affect the folding form of the fused object, but also when GFP is fused with a protein whose self-folding form is not appropriate, the self-folding form of GFP will also be destroyed, resulting in abnormal weak fluorescence
[0007] Therefore, GFP or EGFP is not suitable for fusion expression with various proteins, and when it is fused with most proteins, it often leads to changes in the activity of the corresponding proteins, or changes in the spatial structure, making it difficult to exert the original properties of these proteins , such as binding properties, etc.
So far, no one has achieved the expression and purification of the fusion protein of EGFP and EGF

Method used

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  • Fusion protein of pellicle growing gene and green fluorescence albumen
  • Fusion protein of pellicle growing gene and green fluorescence albumen
  • Fusion protein of pellicle growing gene and green fluorescence albumen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Construction of pET28a-EGFP-EGF vector

[0103] Using the plasmid pEGFP-N1 (purchased from Clontech) with enhanced green fluorescent protein as a template, the EGFP sequence was amplified with primers GFPNde and GFPECOR, then digested with Ndel and EcoR1, and then inserted into the pET28a vector cut by Ndel / EcoR1 (available from Novagen) skeleton, wherein,

[0104] GFPNde (SEQ ID NO: 2):

[0105] 5'-GGGAATTCCATATGGTGAGCAAGGGCGAG-3';

[0106] GFPECOR (SEQ ID NO: 3):

[0107] 5'-CCGGAATTCCTTGTACAGCTCGTCCATG-3'.

[0108] Sequencing identified the vector containing EGFP, which was named pET28a-EGFP. In order to insert coding sequences of other proteins at the 3' end of the EGFP coding sequence, the inventors inserted the following sequence MCSU between the restriction sites EcoRI and XhoI of pET28a-EGFP to introduce BamHI and HindIII restriction sites:

[0109] 5'-AATTCAGCCAGCCGAAGCTTCGCGGATCCTAAC-3' (SEQ ID NO: 4); and

[0110] 5'-TCGAGTTAGGATCCGCGAAGCTTCGGCTGGCTG-3'...

Embodiment 2

[0124] Expression and purification of EGFP-EGF protein

[0125] The identified pET28a-EGFP-EGF plasmid was transformed into the host strain Escherichia coli HMS174 (DE3) (purchased from ATCC) competent cells, and a single colony was picked and inoculated in LB liquid medium containing kanamycin resistance, 37 Cultivate overnight with shaking.

[0126] The next day, dilute 1:20 in 300ml LB liquid medium containing kanamycin resistance, and continue shaking culture to OD 600 About 0.6, add IPTG to make the final concentration 1mM, induce 3h at 30°C. Collect the bacteria by centrifugation, resuspend the bacteria with 30ml PBS, sonicate until the cells are completely lysed, centrifuge at 13000rpm at 4°C for 10 minutes, and collect the supernatant.

[0127]Add Ni-NTA resin solution (purchased from Qiagen) to the protein supernatant, and incubate with rotation at 4°C for 7-8 hours. Transfer the incubation solution to the elution column, wash the resin with 5 times the volume of N...

Embodiment 3

[0138] EGFP-EGF protein binding to cells and endocytosis

[0139] To analyze the endocytosis of EGFP-EGF protein, inoculate 3 × 10 4 Hela cells (purchased from ATCC) were placed in 24-well plates; the next day, the cell culture medium was replaced with serum-free DMEM medium 3 hours before the experiment. After 3 hours of serum starvation, the purified EGFP-EGF protein was added to the cells so that the final concentration of EGFP-EGF was 200 ng / ml, incubated at 37°C for 1 hour, and washed 3 times with PBS at room temperature for 10 minutes each. At the same time, EGFP protein (200ng / ml) was used as a negative control.

[0140] Photographs were taken with a fluorescence microscope (Axioskop 2; Carl Zeiss, Gottingen Germany). In order to prove that EGFP-EGF mediates endocytosis through EGF, a 200-fold excess (mass ratio) of EGF was added to the EGFP-EGF in the competition control group.

[0141] The results are shown in Figure 4, where A is the binding and endocytosis of EGF...

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Abstract

The invention discloses a fusion protein in the biological and biomedical domain, wherein the first part of the fusion protein is green fluorescent protein or its activity segment; the second part is epidermal growing factor or its activity segment; the amino acid connector with fitful length is between two parts. The fusion protein can be applied to study acceptor endocytosis of epidermal growing factor and its molecular elementary and the detection of acceptor expressive level of epidermal growing factor. The invention has good sensitivity during detecting stage without doing photopathic operation, which doesn't display radioactive pollution.

Description

technical field [0001] The invention belongs to the fields of molecular biology and biomedicine; specifically, the invention relates to a fusion protein capable of efficiently binding to epidermal growth factor receptor; the invention also relates to the application of the protein. Background technique [0002] Epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) play an important role in the growth, differentiation and proliferation of many cells in the human body. Epidermal growth factor receptor is highly expressed in many tumor cells and is an ideal target for tumors. The research on the mechanism of endocytosis and signal transduction of epidermal growth factor receptor has always been one of the hot spots in molecular biology and molecular medicine research, and it is also the target of tumor gene therapy and biological therapy. [0003] In the past, isotope-labeled EGF was used to study the endocytosis mechanism of epidermal growth factor recepto...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K14/485
Inventor 李宗海顾健人蒋华徐宇虹
Owner SHANGHAI INST OF ONCOLOGY
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