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Targeted fusion proteins and methods for the characterization of cellular membrane domains

a technology of fusion proteins and cellular membranes, applied in the field of cellular biology and membrane and protein biochemistry, can solve the problems of many of their properties remaining elusiv

Inactive Publication Date: 2006-12-14
RODGERS WILLIAM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides compositions and methods for studying the properties of glycolipid-enriched membrane (GEM) domains in plasma membranes. These domains are enriched in cholesterol and specific proteins, and are involved in cell signaling. The invention involves the use of labeled fusion proteins that contain regions of GEM and non-GEM targeting signals. These fusion proteins can be used to gain better understanding of intracellular trafficking and to screen agents that alter the localization of peptides. The invention also includes the use of fluorescent proteins, such as GFP, for double-labeling studies and energy transfer measurements. Overall, the invention provides new tools for studying GEM domains and their functions in cell signaling.

Problems solved by technology

Despite the current interest in GEM domains, many of their properties remain elusive, due in large in part to the fact that most studies rely on detergent lysis of cells in order to separate the detergent-resistant GEM fraction from the non-GEM fraction of the plasma membrane.

Method used

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  • Targeted fusion proteins and methods for the characterization of cellular membrane domains
  • Targeted fusion proteins and methods for the characterization of cellular membrane domains
  • Targeted fusion proteins and methods for the characterization of cellular membrane domains

Examples

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example 1

Fusion Proteins Construction and Plasma Membrane Localization

[0101] Gene Construction. The separate GFP fusion proteins are outlined in FIG. 1A. Each was constructed using the GFP cloning and expression vector pWay20 (Lo et al., 1998). This vector contains a Sma1 site immediately upstream of and in-frame with enhanced GFP (eGFP, Clontech, Carlsbad, Calif.) and a stop codon at the 3′ end of eGFP. A CMV promoter drives gene transcription.

[0102] The GEM-targeted GFP molecule was constructed using the first 10 amino acids of p56Lck followed by an intermediate four-residue poly-glutamine insert, and finally eGFP itself(L10-GFP) (FIG. 1A). The polyglutamine insert was added to provide flexibility between the adjacent protein domains (Minor and Kim, 1994). The oligonucleotides used for encoding the N-terminal region of p56Lck and the poly-glutamine spacer were: ATGGGCTGTGTCTGCAGCTCAAACCCTGAAAACAAC-AACAAC (coding; SEQ ID NO:1) and GTTGTTGTTGTTTTCAGGGTTTGAGC-TGCAGACACAGCCCAT (noncoding SEQ...

example 2

T Cell Stimulation and Targeting of GEM Domains by Fusion Proteins

[0108] Stimulation of T cells by antibody-mediated crosslinking of the T cell receptor (TcR) results in patching of GEM domains at the site of TcR crosslinking (Janes et al., 1999). Importantly, FIGS. 2A-B shows L10-GFP is selectively enriched in membrane caps that form as a result of crosslinking of the TcR by OKT3-coated bead. For example, each cell shown in FIG. 2A has bound an OKT3-coated bead, and the asterisk indicates this. In the cell expressing L10-GFP, the fluorescence is enriched in the region of the plasma membrane contacting the bead. Conversely, S15-GFP is not enriched in a membrane cap. Actin staining using phalloidin confirmed that a membrane cap was present at the bead contact site in the S15-GFP sample (data not shown). Furthermore, the amount of enrichment of the respective GFP constructs at the bead contact site was quantitated by measuring the fluorescence intensity of the plasma membrane around ...

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Abstract

Cell membranes containing glycolipid-enriched membrane (GEM) and non-glycolipid-enriched membrane (non-GEM) domains are targeted using fusion proteins that are anchored in the cell membrane. Fusion proteins to target GEM (or non-GEM) domains are comprised of a selected fluorescent polypeptide, a membrane-targeting sequence of p56Lck (or pp60c-Src for non-GEM domains) and a linker inserted between the polypeptide and the membrane targeting sequence. Localization of fusion proteins in GEM and non-GEM domains is assessed using techniques including confocal microscopy, fluorescence-based techniques, and membrane fractionation. Using these techniques, compounds are screened for their effect on GEM and non-GEM domains of live cells. These fusion proteins therefore represent useful tools for studying subcellular trafficking and the function of discrete compartments in the plasma membrane.

Description

[0001] This application is related to, and claims a benefit of priority from, copending provisional U.S. Provisional Ser. No. 60 / 304,030, filed Jul. 9, 2001, the entire contents of which are hereby expressly incorporated by reference for all purposes.BACKGROUND OF THE INVENTION [0002] I. Field of the Invention [0003] This invention relates to the fields of cellular biology and membrane and protein biochemistry. In particular, the present invention provides fusion proteins encoding fluorescent proteins. The fusion proteins target glycolipid-enriched membrane (GEM) and non-glycolipid-enriched membrane (non-GEM) domains of cell membranes by virtue of membrane-anchoring signal from p56lck or pp60c-scr, respectively. [0004] II. Background [0005] The plasma membrane of mammalian cells contains domains representing distinct regions within the lipid bilayer with a composition that is different from the bulk composition of the plasma membrane. An important example of such domains are glycoli...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/53C07H21/04C12P21/04C07K14/435C07K14/705
CPCC07K14/705C07K2319/00C07K2319/02G01N33/92C07K2319/60C12N9/1205G01N33/5041C07K2319/033
Inventor RODGERS, WILLIAM
Owner RODGERS WILLIAM