A New Method for Immobilization of His-Tagged Proteins with the Application of Low-Frequency AC Electric Field

Abstract

Continued advancement of protein array, bioelectrode, and biosensor technologies is necessary to develop methods for higher amount and highly oriented immobilization activity of proteins. In pursuit of these goals, we developed a new immobilization method by combining electrostatic transport and subsequent molecular diffusion of protein molecules. Our developed immobilization method is based on a model that transports proteins toward the substrate surface due to steep concentration gradient generated by low-frequency AC electric field. The immobilization of the maximum amounts can be obtained by the application of the AC voltage of 80 Vpp, 20 Hz both for His-tagged Green Fluorescent Protein (GFP) and Discosoma sp. Red Fluorescent Protein (DsRed), used as model proteins. The amounts of the immobilized His-tagged GFP and DsRed were approximately seven-fold higher than that in the absence of the application of low-frequency AC electric field. Furthermore, the positively and negatively charged His-tagged GFP at acidic and alkaline pH were immobilized by applying of low-frequency AC electric field, whereas the non-charged His-tagged GFP at the pH corresponding to its isoelectric point (pI) was not immobilized. Therefore, unless the pH is equal to pI, the immobilization of electrically charged proteins was strongly enhanced through electrostatic transport and subsequent molecular diffusion.

Predicting the Orientation of Adsorbed Proteins Steered with Electric Fields Using a Simple Electrostatic Model

“…of proteins based on the natural heterogeneous distribution of charges, , by performing chemical modifications on either the −NH 2 or the −COOH terminal groups, , or by directed mutations . Alternatively, and although it can only be applied to conductive materials, the adsorption process can also be influenced by the application of an external electric field, − potentially affecting the orientation of such proteins. − …”

Research progress of multi-enzyme complexes based on the design of scaffold protein

“…The use of affinity tags to immobilize functional proteins on solid carriers is an attractive method in protein microarray technology (Freitas et al 2022 ). For, example, materials such as glass and acrylic glass functionalized with nickel (II) ions have also been employed in affinity-mediated oriented immobilization of His-tagged recombinant proteins (Kulsharova et al 2018 ; Takahashi et al 2018 ). Among the commonly used fusion protein tags, glutathione S-transferase (GST) proteins have been indispensable tools for protein–protein interaction studies and have extensive applications in recombinant protein purification and reversible protein immobilization.…”

Tag-mediated single-step purification and immobilization of recombinant proteins toward protein-engineered advanced materials

“…Materials such as glass and acrylic glass functionalized with nickel (II) ions have also been employed in affinity-mediated oriented immobilization of His-tagged recombinant proteins [41] , [127] . However, despite the higher cost and limited availability, inert metals such as gold are still preferable for biomolecule-oriented immobilization due to their stability, biocompatibility, ability for bioconjugation and enhanced physicochemical properties (e.g., optical, electronic and catalytic) [124] , [128] , [129] , [130] .…”