New Developments for the Site-Specific Attachment of Protein to Surfaces

Abstract

Protein immobilization on surfaces is of great importance in numerous applications in biology and biophysics. The key for the success of all these applications relies on the immobilization technique employed to attach the protein to the corresponding surface. Protein immobilization can be based on covalent or noncovalent interaction of the molecule with the surface. Noncovalent interactions include hydrophobic interactions, hydrogen bonding, van der Waals forces, electrostatic forces, or physical adsorption. However, since these interactions are weak, the molecules can get denatured or dislodged, thus causing loss of signal. They also result in random attachment of the protein to the surface. Site-specific covalent attachment of proteins onto surfaces, on the other hand, leads to molecules being arranged in a definite, orderly fashion and uses spacers and linkers to help minimize steric hindrances between the protein surface. This work reviews in detail some of the methods most commonly used as well as the latest developments for the site-specific covalent attachment of protein to solid surfaces.

Interfacing "Soft" and "Hard" Matter with Exquisite Chemical Control

“…Most methods used for chemoselective immobilization of proteins are based on ligation methods originally developed for the synthesis, semi-synthesis, and selective derivatization of proteins by chemical means (see Table 1) [16]. All these methods involve the derivatization of a protein with a unique chemical group at a defined position, which will later react chemoselectively with a complementary group previously introduced into the inorganic surface.…”

“…The use of the Cys thiol group is one of the most common method used by the scientific community for achieving ordered immobilization of different proteins and viruses onto inorganic supports (reviewed in detail in [16]). It should be noted, however, that is not general method and can only be applied when there is only one reactive Cys residue, either artificially introduced or naturally occurring, on the protein to be attached.…”

“…The reaction between these two groups should be highly chemoselective, thus behaving like a molecular 'Velcro' [15,16]. Finally, the use of hydrophilic spacers and linkers may also help to minimize the potentially detrimental interaction between the protein and the inorganic support.…”

Recent developments in the site‐specific immobilization of proteins onto solid supports

“…3 The use of recombinant affinity tags addresses the orientation issue. 2,[16][17][18][19][20][21][22][23][24][25] However, in most cases, the interactions of the tags are reversible and not stable over time. 24,[26][27][28][29] Site-specific covalent immobilization, on the other hand, allows the proteins to be arranged in a definite, controlled fashion.…”

“…The reaction between these two groups should be highly chemoselective, thus behaving like a molecular ''velcro.'' 22,30 Also, the reaction should work efficiently under physiological conditions (i.e., in aqueous buffers around neutral pH) to avoid the denaturation of the protein during the coupling step. Finally, it is desirable that the reactive group on the protein was obtained using recombinant protein expression techniques.…”