Gfp fusion proteins and their use

a technology of fusion proteins and fusion proteins, applied in the field of gfp fusion proteins and their, can solve the problems of difficult to prove and not give results reflective of the natural sta

Inactive Publication Date: 2012-06-07
THE BOARD OF TRUSTEES OF THE UNIV OF ILLINOIS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015]4. The use of green fluorescent protein (GFP) in the study of cellular signaling allows not only the observation of G protein trafficking, but the opportunity to study the dynamics of G proteins in real time as well as their function.

Problems solved by technology

G proteins may leave the membrane in response to neurotransmitter or hormone signals, but this has been very difficult to prove.
However, this attachment may alter the function of the protein fused with GFP consequently may not give results reflective of the natural state.

Method used

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  • Gfp fusion proteins and their use

Examples

Experimental program
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Embodiment Construction

[0029]Construction of Gαs-GFP

[0030]Full length cDNAs encoding Gαs were excised from the PcDNA-1 vector by digesting with Sam I and Xba I restriction enzymes. The full length EGFP cDNA was obtained by PCR from the PEGFP-N3 using appropriate primers (sense 5′ GGAATTCATGAGCAAGGGCGAGGAACTG-3′ (SEQ ID NO: 8); antisense 5′-GCTCTAGACGACTTGTACAGCTCGT-3′) (SEQ ID NO: 9) and adding restriction sites to its cDNA (EcoR I at the initiation codon and Xba I at end of cDNA). To insert the EGFP within the sequence of Gαs, the first fragment of Gαs (from 1 to 71 amino acids) was amplified by PCR with restriction sites for Kap 1 at initiation codon and EcoR I at end of the fragment. The cDNA of the fragment was cloned into PcDNA3 vector by the Kap 1 and EcoR 1 restriction sites using primers (sense 5′GGGTACCATGGGCTGCCTCGGCAACA-3′ (SEQ ID NO: 10); antisense 5′-GGAATTCGTCCTCTTCGCCGCCCTTCT-3′) (SEQ ID NO: 11). Modified 7 EGFP cDNA was spliced into the first fragment of Gαs by EcoR 1 and Xba 1 restriction...

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Abstract

The present invention provides fusion proteins including a green fluorescent protein inserted into the internal amino acid sequence of a Gαs protein and further provides method of using the fusion protein construct to follow activation of a G-protein receptor by a candidate drug.

Description

[0001]This application is a Continuation of co-pending U.S. patent application Ser. No. 10 / 482,980, filed Sep. 22, 2004, which is a national stage application of PCT / US02 / 21484 filed Jul. 3, 2002, which claims priority to U.S. Provisional Application No. 60 / 303,622 filed Jul. 6, 2001. The disclosures set forth in the above-referenced applications are incorporated herein by reference in their entireties.[0002]This invention was made with government support under MH39595-10 and AG15482 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.[0003]The present invention relates a protein that is constructed by adding a green fluorescent protein designated GFP that is internal to the amino acid sequence of a G protein, in particular the Gαs protein. The resulting fusion protein is a non-radioactive marker used, for example, for high throughput screening of G protein-coupled receptor drug targets.BACKGROUND OF THE INVENTION[0004]A family of h...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12P21/02C07K19/00C07K14/435
CPCC07K14/43595C07K14/4722G01N2333/726G01N33/76C07K2319/60
Inventor RASENICK, MARKYU, JIANG-ZHOU
Owner THE BOARD OF TRUSTEES OF THE UNIV OF ILLINOIS
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