Foreign Gene Expression in Photosynthetic Bacteria

Abstract

Large-scale production of membrane proteins in functional form is an arduous task. The overexpression of membrane proteins is fraught with unique challenges, and systems presently in use often fail to generate products which are compatible with subsequent solubilization, stabilization, and purifi cation steps. To address these requirements, it is possible to exploit the unique physiology of the Rhodobacter species of photosynthetic bacteria, which produces extremely large quantities of internal membranes (invaginations of the cytoplasmic membrane) under certain growth conditions in response to changes in light intensity and/or oxygen tension. Towards this end, an expression system has been designed that coordinates synthesis of foreign membrane proteins with synthesis of new membrane into which they can be incorporated. These intracytoplasmic membrane (ICM) vesicles sequestering the newly synthesized foreign proteins are readily isolated by differential centrifugation following cell lysis. A diverse set of foreign membrane proteins – spanning a range of isoelectric points, molecular weights and predicted membrane topologies and representing several prokaryotic and eukaryotic species – has been expressed heterologously in Rhodobacter. Many target membrane proteins can be produced and purifi ed in a semi-automated fashion at levels greater than 10 mg per liter of culture. This expression system is versatile, offering many strategies to achieve success in expression of membrane proteins. However, it is not necessarily restricted to the production of membrane proteins. For instance, Rhodobacter possesses advantages for the expression of soluble proteins that require an abundance of membrane surface (e.g., proteins only peripherally- or transiently-associated with the membrane) or proteins requiring complex redox cofactors which are native to Rhodobacter. In addition, Rhodobacter ICMs are easily isolated and are naturally enriched in target membrane proteins; thus, these membranes also facilitate biochemical studies, making this expression system valuable for a wide range of applications.