A new system for heterologous expression of membrane proteins: Rhodospirillum rubrum

Abstract

a b s t r a c t Heterologous expression of membrane proteins has met with only limited success. This work presents a new host/vector system for the production of heterologous membrane proteins based on a mutant of the facultatively phototrophic bacterium Rhodospirillum rubrum. Under certain growth conditions, R. rubrum forms an intracytoplasmic membrane (ICM) that houses the photosynthetic apparatus, the structural proteins of which are encoded by puhA and pufBALM. The mutant R. rubrum H2, which was constructed by allelic exchange deleting puhA and pufBALM, does not form ICM. This strain was used as a host for a plasmid expressing the Pseudomonas aeruginosa membrane protein MscL from the Rhodobacter capsulatus puc promoter. ICM was formed in the H2 strain producing MscL but not in the vector control strain. These results suggest that a heterologous membrane protein stimulates ICM formation in R. rubrum and indicate that the capacity to form an ICM that can accommodate heterologous proteins makes R. rubrum a host that will be useful for membrane protein production. P. aeruginosa MscL, which forms inclusion bodies when produced in Escherichia coli, was expressed in R. rubrum H2 and purified from membranes with a yield of 22.8-23.4 mg/L culture (5.53-5.60 mg/g cell paste). Additionally Streptomyces lividans KcsA and P. aeruginosa CycB were produced and purified from R. rubrum H2 with yields of 13.7-14.4 mg/L culture (2.19-2.55 mg/g cell paste) and 6.6-7.4 mg/L culture (1.1-1.2 mg/g cell paste), respectively. Ó 2009 Elsevier Inc. All rights reserved. Introduction Overproduction of membrane proteins in heterologous systems has met with only limited success. While Escherichia coli is very useful for the production of many soluble proteins, expression of membrane proteins in this host may be lethal or result in the production of inclusion bodies formed by misfolded proteins. As a result of the difficulties in the production of membrane proteins in currently available systems, structural studies of membrane proteins have lagged significantly behind such studies of soluble proteins. A new bacterial system for the production of membrane proteins would provide the opportunity to expand the study of this important class of proteins. Rhodospirillum rubrum is a bacterium that is capable of phototrophic growth under anaerobic conditions in the light and chemotrophic growth under aerobic conditions in the dark. Under reduced oxygen conditions, R. rubrum forms an intracytoplasmic membrane (ICM) 2 that houses the photosynthetic apparatus. This apparatus consists of the light-harvesting antenna and the photochemical reaction center. Aerobically grown cells use aerobic respiratory metabolism and lack the photosynthetic apparatus and ICM. The proteins of the pigment-protein complexes in the ICM are encoded by puhA and pufBALM. Knockout mutants deleted in either puhA or pufBALM lack ICM, but ICM formation is restored by complementatio