Solid Binding Peptides: Applications in Affinity Protein Purification and Bioprotonic Devices

Abstract

Solid binding peptides (SBPs) are short sequences of amino acids that can be fused to proteins to endow them with the ability to bind to inorganic or synthetic interfaces without affecting structure or function. Here, we describe the use and optimization of SBP usage in two important technological applications: the affinity purification of proteins, and the construction of a new class of bionanoelectronic devices. First, we use Car9, a silica binding peptide whose interaction with silica can be disrupted with lysine, to optimize the conditions for the purification of Car9-tagged proteins using underivatize silica. We demonstrate that capture of Car9 fusion proteins is enhanced on small particle and large pore size silica gels, that addition of 0.3% Tween 20 prevents non-specific protein binding and that efficient elution is achieved under alkaline conditions. Using the optimized conditions, we developed a small-scale purification kit that allows the inexpensive recovery of milligram-quantities of Car9-tagged proteins with purities higher than 90%. Then, we extended the functionality of SBPs to the integration of rhodopsins to electronic devices for the light activated conversion of protonic currents into electronic currents. To this end we fused Pd4, a Pd-binding peptide, to the N-terminus of the green light absorbing H.turkmenica deltarhodopsin (HtdR) to bring the protein and protons transported by it to a closer proximity to the surface of Pd/PdHx-based devices. We demonstrate that addition of the Pd4 tag leads to a nearly one order of magnitude increase in electronic signal when compared to untagged protein. To enhance their functionality and develop multi-wavelength devices, we optimized the production of a blue absorbing proteorhodopsin (BPR) by exploring signal sequence requirements and export pathway. We show that BPR does not rely on the Sec pathway for inner membrane integration and that its signal sequence is not necessary to obtain a functional product. We also identified a mutation in the signal peptidase I recognition site that prevents cleavage of the signal sequence resulting in a product that exhibits all functional attributes of the wild type protein but produced at higher levels. After optimizing the production of BPR, we constructed a Pd binding BPR to fabricate HtdR- and BPR-based devices with light absorption maxima - and consequently photocurrent maxima - separated by 37 nm. These devices exhibit wavelength-dependent photocurrent production, opening up the possibility to develop a new type of biological camera. Together, this work demonstrates that SBPs are powerful tools for the development of cost effective purification schemes and more efficient devices.