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Target sequences for synthetic molecules

a synthetic molecule and target sequence technology, applied in the field of synthetic molecules, can solve the problems of labeling stoichiometry, inability to perform a large number of cells, and often encountered disruption of biological activity,

Inactive Publication Date: 2005-08-11
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is based on the discovery that certain molecules called biarsenical molecules can react with specific amino acid sequences, called target sequences, to label polypeptides containing those sequences. This allows for the stable expression of polypeptides containing a target sequence and the observation of protein-protein interactions and conformational changes in proteins. The invention also provides vectors for introducing a target sequence into a cell and methods for labeling a carrier molecule. The technical effects of the invention include improved methods for labeling polypeptides and the ability to observe protein-protein interactions and conformational changes in proteins.

Problems solved by technology

Using this approach, problems of labeling stoichiometry and disruption of biological activity are frequently encountered.
These processes can be tedious and typically cannot be performed on a large population of cells.
However, these chemical labels are promiscuous.
However, GFP is limited in versatility because it cannot reversibly label the polypeptide.
GFP's large size frequently perturbs the protein interest upon binding.
Although red emitting fluorescent proteins are known to the art, their development has been slow and their utility has been greatly restricted.

Method used

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  • Target sequences for synthetic molecules
  • Target sequences for synthetic molecules
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Examples

Experimental program
Comparison scheme
Effect test

example 1

Target Sequence Generated on AcpS

[0249] A target sequence that includes the SlyD (SEQ. ID NO: 4) tetracysteine sequence, CCGGKGNGGCGC (SEQ. ID NO: 5) was introduced onto the Carboxy-terminus of Acyl Carrier Protein S (AcpS). Since AcpS has only one endogenous cysteine amino acid and since AcpS is a robust stable protein, a substitution at the Carboxy-terminus could be made without altering the solubility of the properly folded protein. The four cysteines comprising the SlyD tetracysteine sequence were introduced at the carboxy-terminus of the protein as seen in SEQ. ID NO: 6. The mutated AcpS is referred to as AcpS+4Cys. The substitutions were generated using polymerase chain reaction with primers specific for the encoding the expression of the desired tetracysteine sequence. The nucleic acid sequence encoding the cysteine substituted AcpS was inserted into the pRSET vector (Invitrogen, Carlsbad, Calif., Catalog # V351-20) using restriction sites inherent to the vector's multiple c...

example 2

Binding Modes for Biarsenical Molecules to Target Sequences

[0251] The mode of binding of a biarsenical to a target sequence was examined using the Expressway™ in vitro protein synthesis kit (Invitrogen, Carlsbad, Calif.) and SDS-PAGE. Following the manufacture's protocol 1 μg of SlyD+His tag (SEQ. ID NO: 8), SlyD-C167A / C168A (SEQ. ID NO: 9), and SlyD-trunc171 (SEQ. ID NO: 10) vector DNAs were added to a total volume of 50 μL of S30 E. coli extract and reaction buffer. The reaction was placed at 37° C. with 225 rpm shaking for two hours. After incubation 5 μL of RNase A was added to the reaction, after which an additional 15 minute incubation at 37° C. was performed. Protein from the in vitro protein synthesis reaction was prepared for SDS-PAGE analysis through an acetone precipitation procedure. 5 μl of reaction was added to 20 μL of 100% acetone. After mixing well the acetone solution was centrifuged for 5 minutes at room temperature in a microcentrifuge at 12,000 rpm. The superna...

example 3

Specificity of Biarsenical Molecules for Tetracysteine Sequences

[0253] To demonstrate specificity of biarsenical compounds for different tetracysteine sequences several chimeric proteins were constructed. The native SlyD sequence (SEQ. ID NO: 4) was cloned into the pRSET vector (Invitrogen, Carlsbad, Calif.) using standard molecular biology techniques. Purified protein was produced from this vector by first transforming BL21 (DE3) cells (Invitrogen, Carlsbad, Calif., Catalog # C6010-03) and plated on LB-ampicillin plates. A single colony was selected and grown in one liter of liquid LB broth to a density of 1 O.D. and 1 mM IPTG was added to induce protein expression. After three hours of protein induction the culture was harvested by centrifugation at 10,000×g for 5 minutes at 4° C. The cell pellet was resuspended in 50 mM HEPES (pH 7.5), 140 mM NaCl and sonicated on ice for a total of two minutes. The E. coli lysate was separated by centrifugation at 25,000×g for 20 minutes at 4° ...

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Abstract

The invention is based on the discovery that certain biarsenical molecules react with specified target sequences, thereby providing a facile means for labeling polypeptides containing the target sequence. The invention is useful in creating stable mammalian cell lines expressing a certain tetracysteine tagged polypeptides, thereby overcoming toxicity associated with native tetracysteine. In addition, the invention allows for orthogonal labeling of polypeptides, thereby allowing for the observation of protein-protein interactions and conformational changes in proteins, for example.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 60 / 513,031, filed Oct. 22, 2003, the disclosure of which is incorporated by reference herein in its entirety.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] This invention relates generally to compositions and methods for labeling molecules, and more specifically to small, synthetic molecules that react with target sequences. [0004] 2. Background Information [0005] Many techniques in the biological sciences require attachment of labels to molecules, such as polypeptides. For example, the location of a polypeptide within a cell can be determined by attaching a fluorescent label to the polypeptide. [0006] Traditionally, labeling has been accomplished by chemical modification of purified polypeptides. For example, the normal procedures for fluorescent labeling require that the polypeptide be covalently reacted in vitro with a fluorescent dye,...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07F9/70C07H21/04C07K7/08C07K16/18C12NG01N33/53G01N33/532
CPCG01N33/532
Inventor HANSON, GEORGE
Owner LIFE TECH CORP
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