Membrane proteins (MP) are difficult to study because of their hydrophobicity and tendency to aggregate when removed from their aqueous environment. MP must be isolated by first disrupting the cell and harvesting the cell membranes using ultracentrifugation. However, this process is labor intensive and can result in proteolysis of membrane proteins. In this study, we will evaluate methods of cell disruption, in order to optimize the extraction of the MP Anabaena Sensory Rhodopsin (ASR). ASR is a seven transmembrane helical protein with a covalently attached retinal cofactor. The protein undergoes a photochromic shift that fulfills its role as a photochromic sensor for the cyanobacterium. However the molecular details are not well understood. To enable such studies large amounts of active protein are needed. Here we will optimize the protein extraction method. We will utilize both mechanical cell disruption with sonication or shearing, and chemical disruption using detergents. The type of detergent can also impact the extraction efficiency and protein activity, thus a set of detergents including triton-X100, tween-20, and dodecyl-maltoside will be explored. We will evaluate both the yield of the protein and the quality of the protein by gel electrophoresis and UV-visible spectroscopy. Preliminary results have identified the optimal cell density for induction as O.D. range 0.5-0.6 at 600 nm which maximizes cell pellet production and protein folding visualized by characteristic color development. Future work will optimize the extraction, facilitating studies of ASR function by embedding ASR in protein lipid nanodiscs.

Progress Toward an Efficient Method for the Extraction of the Membrane Protein Anabaena Sensory Rhodopsin

ACS Spring 2022

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