Abstract
Microbial rhodopsins (M-Rho) are found in Archaea, Bacteria and some species of Eukarya and serve as light-driven ion pumps or mediate phototaxis responses in various biological systems. We previously reported an expression system using a highly expressible mutant, D94N-HmBRI (HEBR) from Haloarcula marismortui, as a leading tag to assist in the expression of membrane proteins that were otherwise difficult to express in E. coli. In this study, we show a universal strategy for the expression of two M-Rho proteins, either the same or different types, as one fusion protein with the HEBR system. One extra transmembrane domain was engineered to the C-terminal of HEBR to express another target M-Rho. The average expression yield in this new system reached a minimum of 2 mg/L culture, and the maximum absorbance of the target M-Rho remained unaltered in the fusion forms. The fusion protein showed a combined absorbance spectrum of a lone HEBR and target M-Rho. The function of the target M-Rho was not affected after examination with functional tests, including the photocycle and proton pumping activity of fusion proteins. In addition, an otherwise unstable sensory rhodopsin, HmSRM, showed the same or even improved stability under various temperatures, salt concentrations, and a wide range of pH conditions. This HEBR platform provides the possibility to construct multi-functional, stoichiometric and color-tuning fusion proteins using M-Rho from haloarchaea.
Highlights
In addition to unveiling the new functions and biophysical properties of those Microbial rhodopsins (M-Rho) proteins, efforts were exerted to explore their various potential applications
We successfully extended one transmembrane segment from highly expressible bacteriorhodopsin (HEBR) to fuse with various target M-Rho proteins, including Halobacterium salinarum bacteriorhodopsin (HsBR), Natronomonas pharaonis halorhodopsin (NpHR), Haloarcula marismortui sensory rhodopsin II (HmSRII), and Haloarcula marismortui sensory rhodopsin M (HmSRM)
The yield of HEBR alone reached 60–70 mg/L in culture without any reconstitution or refolding[24], and the maximum absorbance (Abs-max) of HEBR26 was 552 nm, almost identical to the wild type protein, HmBRI. Such an overexpression yield in E. coli system inspired the adoption of HEBR as a co-expression tag for other kinds or types of M-Rho proteins to create fusion proteins featuring multi-capability yet with ample expression yield for further creative applications
Summary
In addition to unveiling the new functions and biophysical properties of those M-Rho proteins, efforts were exerted to explore their various potential applications. The unique properties of BR, their photochromism and high thermal stability, have been the primary features in physicochemical studies. The versatility of other M-Rho proteins was shown in recent developments Both halorhodopsin (HR), a light-driven inward chloride pump, and light-gated inward cation channel ChR2 were shown to deactivate and activate, respectively, nerve fiber under different wavelengths of light[22]. To create even more versatile applications, expressing different types of M-Rho as one fusion protein is a logical approach, since it will enable the design of new, multi-capability proteins, which can be fixed at a desired stoichiometric ratio of component moieties. The optical property, function and stability of the target M-Rho protein were examined and found to be intact or unaltered
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