Site Specific Protein Immobilization via Bioorthogonal Reactions

RAJESH VISWANATHAN; GUILLERMO R. LABADIE; C. DALE POULTER

University, Durham, NC, USA

Simposio; 40th National Organic Chemistry Symposium; 2007

American Chemical Society, Organic Division

Abstract: Immobilization of proteins in a microarray format that preserves their native tertiary structure and activity is a challenging task. Our efforts toward this goal involves interception of protein farnesylation with designed analogs leading to a general method for achieving site specific tagging of proteins. Use of an azido and an alkyne derived FPP analog leads to tagged proteins capable of engaging in ‘click’ chemistry. A set of four commonly used proteins namely : Green Fluorescent Protein (GFP), Maltose Binding Protein (MBP), Glutathione S-Transferase (GST) and -Galactosidase (-Gal) are chosen to demonstrate the effectiveness of the strategy. Modified proteins are characterized by ES-MS. Glass slides that are suitably modified bearing complimentary functionality assist in covalent immobilization via Cu-catalyzed Huisgen [3+2] cycloaddition (Click) reaction. Surface immobilized proteins are detected using fluorescently labeled antibodies. As a proof of principle, current efforts demonstrate preservation of enzymatic activity after immobilization on glass slide for GST.