Method of site specific labeling of proteins and uses therefor
AssigneeSmithKline Beecham Corporation
InventorsWenfang Chen, Thomas Meek, David Powell, David Tew
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Abstract
Methods for site-specific modification of protein are provided. These methods modify proteins which have been labeled at a particular site by the reaction of a transglutaminase with a glutamine peptide sequence which has been engineered into the protein. The site-specific modification methods of the invention are useful for producing reagents useful in high throughput screening methods and in producing protein delivery vehicles for specifically targeting cellular and non-cellular targets. Also described are improved biotinylation reagents.
Claims38
A method of screening for a candidate compound which interacts with a first protein, comprising the steps of:
- modifying a first protein to contain the sequence Gln-Ser-Lys-Val-(Leu or lie), SEQ ID NO:
- 1;
- labeling said modified first protein by reacting a transglutaminase with said modified first protein and a detectable labeling compound;
- contacting said labeled modified first protein with at least one candidate compound;
- and
- detecting said label, thereby identifying the interaction of said first protein and said candidate compound.
The method according to claim I wherein said method further comprises the steps of:
- replacing said labeling compound on said first protein with a second labeling compound.
A method for site specific labeling of a selected protein comprising the steps of:
- modifying a selected protein to contain a sequence comprising Gln-Ser-Lys-Val-(Leu or Ile), SEQ ID NO:1;
- and
- reacting a transglutaminase with said selected protein and a labeling compound, thereby labeling said protein with said labeling compound at the site of said glutamine residue.
A biotinylation reagent having the formula Biotin-R1-R2, wherein X is a spacer compound and R2 is a compound comprising at least four methylene groups and a NH2 group.
A site specific labeled protein comprising an artificial amino acid sequence:
- (Aa)n-Glnp-Ser-Lys-Val-(Leu or Ile)-(Aa)n′SEQ ID NO:3, wherein n and n′ are independently selected from 0 to 100, and P is a site specific labeling compound.
A molecule comprising a site specific modified protein delivery vehicle comprising an artificial amino acid sequence:
- (Aa)n-Gln-Ser-Lys-Val-(Leu or Ile)-(Aa)n′SEQ ID NO:3, wherein n and n′ are independently selected from 0 to 100, and a moiety to be delivered to a target by the modified protein delivery vehicle.
Dependencies are inferred from the claim text.