UCLA investigators from the Department of Molecular and Medical Pharmacology have developed a system that allows the metabolic biotinylation of secreted proteins in vivo by biotin ligase, both secreted and ER-retained. This process is an improvement over traditional in vitro chemical methods, and does not alter biological functions and reduces the chance of protein degradation.
Biotin (vitamin H) is an essential coenzyme that is also used to tag proteins for detection, labeling, and purification purposes. The process of adding biotin to proteins is called biotinylation. Biotin labeling has also been applied to drug targeting and viral gene therapy vector-targeting strategies. Traditionally, biotin labeling has been performed in vitro by chemical methods. The problem with these chemical methods is that the random and heterogeneous modifications can lead to the inactivation of biological function after mixing with streptavidin or avidin. Antibody biotinylation especially leads to heterogeneous conjugates. Therefore, there is a need for a method that will uniformly biotinylated proteins without altering binding properties and resulting in loss of affinity.
UCLA researchers have developed a system that allows metabolic biotinylation of secreted proteins in vivo by both versions of biotin ligase (secreted and ER-retained). The system is a general approach for production of site-specific biotinylated proteins for streptavidin/avidin-biotin technology. The biotinylation technology also shortens and streamlines the lengthy process of traditional methods and reduces the chance of protein degradation during in vitro biotinylation by exogenous ligase. Not only is this system a significant improvement over traditional chemical methods, the system produces uniformly biotinylated proteins without altering biological functions or binding specificity. Researchers also generated a stable cell line expressing ER-retained BirA, the biotin protein ligase of E. Coli
Investigators have tested the system to biotinylated proteins in vivo by biotin ligase.
|United States Of America||Issued Patent||8,043,830||10/25/2011||2008-501|
system, process, biotinylated, biotin, vitamin H, coenzyme, ligase, protein, antibody, streptavidin, avidin, stable cell line