ABSTRACT
Figure 12.1 Cartoon representation of chemical strategies for protein immobilization yielding random or uniform, oriented proteins at the surface, either through covalent or through noncovalent bond formation. For each case a chemical example is represented. The access to methods for selectively incorporating azide groups into recombinant proteins [22] makes it possible to use the azide for protein immobilization through either Staudinger ligation [23, 24], which requires a phosphine-ester or thioester at the surface, or the Huisgen 1,3-dipolar cycloaddition [25], which requires an alkyne-functionalized surface. Other cycloaddition reactions have been
reported for immobilizing proteins, such as the click sulfonamide reaction (between sulfonyl azides and alkynes) [26] and the DielsAlder reaction (between a diene and an alkene) [27]. The specificity of the immobilization was demonstrated for different proteins such as ribonuclease A, streptavidin (SAv), and Ras proteins. Very recently, the thiol-ene click reaction was employed for the light-induced immobilization of proteins with a genetically encodable CAAX-tag combined with S-farnesylation in cells [28]. The photochemical thioether bond formation between an olefin of the isoprenoid and thiol-functionalized surfaces allowed the immobilization of isolated proteins or proteins directly from expression lysate [29]. In this chapter noncovalent immobilization methods that lead to site-selective orientation of proteins are presented. Supramolecular methods satisfy important requirements of protein arrays preparation, such as the use of mild reagents, buffered conditions, bio-orthogonality, specificity, stability, and reversibility and thus error correction during assembly, better packing, and faster preparation of the protein array. 12.2 Site-Selective Noncovalent Immobilization
Protein semisynthesis by expressed protein ligation (EPL), which involves the chemoselective addition of, for example, a peptide to a recombinant protein, offers further options for covalent attachment of proteins to surfaces, producing a native peptide bond. Through EPL, site-specifically C-terminal thioester-activated proteins have been directly immobilized on N-terminal cysteine-functionalized slides [30]. Camarero et al. proposed a traceless ligand strategy using protein trans-splicing (Fig. 12.2). In this case, the intein domain is split into two fragments (N-intein and C-intein) [31]. The C-intein fragment is covalently immobilized onto a glass surface, while the N-intein fragment is fused to the C-terminus of a protein that is to be attached to the surface. When both intein fragments interact, they form an active intein domain that binds the protein to the surface while releasing the split intein into the solution, yielding a native
peptide bond between the surface and the protein of interest. The naturally split DnaE intein possesses C-and N-intein fragments able to self-assemble spontaneously (K – 105 M-1). Intein-fused maltose-binding protein (MBP) and fluorescent proteins were immobilized, and their (immuno)analysis showed no significant loss of fluorescence, proving the retention of protein conformation [31]. Control experiments with proteins lacking the intein fusion showed no protein attachment.
