Site-Specific, Covalent Attachment of Proteins to a Solid Surface

Abstract

Immobilized and site-specifically labeled proteins are becoming invaluable tools in proteomics. Here, we describe a strategy to attach a desired protein to a solid surface in a covalent, site-specific manner. This approach employs an enzymatic posttranslational modification method to site-specifically label a target protein with an azide; an alternative substrate for protein farnesyl transferase containing an azide group was developed for this purpose. A bio-orthogonal Cu(I)-catalyzed cycloaddition reaction is then used to covalently attach the protein to agarose beads bearing an alkyne functional group. We demonstrate that both the azide incorporation and the capture steps can be performed on either a purified protein target or on a protein present within a complex mixture. This approach involves the use of a four-residue tag which is significantly smaller than most other tags reported to date and results in covalent immobilization of the target protein. Hence it should have significant applicability in protein science.

Vinyl functionalized silica hybrid monolith-based trypsin microreactor for on line digestion and separation via thiol-ene “click” strategy

“…The convective mass transfer process can accelerate the diffusion of compounds, especially biopolymers, to access the monolith surface. Covalent immobilization of enzyme on a capillary monolithic column is an useful method to prepare enzyme microreactor, e.g., immobilization by genetic engineering [24], Staudinger ligation reaction [25], or azide-alkyne click reaction [26]. The immobilized trypsin microreactor by coupling with RPLC-MS/MS, CE and CE-MS has been applied for analysis of proteins and peptides [27][28][29][30].…”

Click Chemistry: Versatility and Control in the Hands of Materials Scientists

“…[46] On the other hand, the Huisgen 1,3-dipolar cycloaddition reaction was used in a novel strategy for covalent attachment of desired proteins to solid surfaces. [47] Azides were attached to the protein via enzymatic posttranslational modification at the C-terminus, which then enabled further immobilization onto alkyne-functionalized agarose beads. In addition, the Huisgen cycloaddition reaction was employed for the development of an efficient system for the immobilization of complex azide-saccharides to microtiter plates via lipid alkyne tethers.…”

Click Chemistry: A Powerful Tool to Create Polymer‐Based Macromolecular Chimeras

“…The copolymer p(LA-co-MPC) was electrospun into continuous fibers with a regular average diameter of ca 2-4 mm. In parallel, Testisspecific protease 50 (TSP50), a breast cancer sensitive protease, was non-specifically functionalized with azide groups [110][111][112] and subjected to a copper (I) catalyzed click chemistry cycloaddition with the p(LA-co-MPC) copolymer. The TSP50-immobilized fibers were successively incubated with anti-TSP50 antigen and then with HRP-Ig.…”

“…A general method for the site-specific covalent conjugation of proteins onto agarose gels was reported by Distefano and coworkers. [111] This approach employed initially an enzymatic post-translational modification method to site-specifically label a target protein with an azide, which was followed by a bioorthogonal Cu(I)-catalyzed cycloaddition reaction to covalently attach the protein to agarose and beads bearing alkyne functional group. It is important to note that in this study it was also demonstrated that the click capture step could be efficiently performed on both purified and complex protein mixtures.…”