Reversible, site‐specific immobilization of polyarginine‐tagged fusion proteins on mica surfaces

Abstract

A large variety of genes is expressed as fusion proteins for the purpose of characterization and purification in molecular biology. We have used this strategy to append polyarginine peptides in order to achieve specific binding of the Arg-tag to atomically flat, negatively charged mica surfaces. We show that the model protein, hexaarginine-tagged green fluorescent protein (GFP), binds to mica via its Arg-tag based on ion exchange of naturally occurring potassium cations. Only non-specific binding was observed with the control protein that is free of the Arg-tag. This novel technology will be widely applicable to orient functional proteins on flat surfaces.

Molecular Cloning and Characterization of a (Lys)6-Tagged Sulfide-Reactive Hemoglobin I from Lucina pectinata

“…The poly-histidine tail [5–8] (His-tag) is the most commonly used to purify and immobilize proteins on numerous surfaces [7] through a non-covalent, reversible immobilization [9]. On the other hand, tags of repeated polar amino acids such as aspartic acid, lysine [8, 10–13], and arginine [14–16] are suitable for covalent immobilization. In fact, we have employed the His-tag technology to study the interaction of hemoglobin I (HbI) from Lucina pectinata with the novel H 2 S signaling molecule.…”

From Protein Features to Sensing Surfaces

“…To achieve immobilization based on electrostatic forces and charged amino acids, the addition of negatively or positively charged stretches of amino acids to one terminus of the protein might be a valuable yet reversible approach [ 64 , 65 ]. A positively charged polyarginine tag was attached to the green fluorescent protein that could be immobilized reversibly to mica, glass, and silica surfaces with no loss of activity [ 64 , 66 ].…”

Engineered (Lys)6-Tagged Recombinant Sulfide-Reactive Hemoglobin I for Covalent Immobilization at Multiwalled Carbon Nanotubes

“…Another technique for the immobilization of proteins is the use of fusion proteins with an extension of charged amino acids, such as polyarginine, , polyhistidine, ,− polyaspartic, and polylysine , tails. The polyhistidine tail (His-tag) is the most commonly used method to purify and immobilize proteins on numerous surfaces but through a noncovalent, reversible immobilization.…”