ABSTRACT
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 (1.1-1.2mg/g cell paste), respectively.
Subject(s)
Bacterial Proteins/genetics , Membrane Proteins/genetics , Rhodospirillum rubrum/metabolism , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Cloning, Molecular , Membrane Proteins/isolation & purification , Membrane Proteins/metabolismABSTRACT
The light-harvesting antenna (LH) proteins of Rhodospirillum rubrum are encoded by pufA and pufB with C-terminal extensions not present in the mature proteins. Point mutations were introduced into pufB, pufA, and both pufB and pufA so that proteins equivalent to the mature proteins were encoded. The mutants with these truncated proteins produced 3-30% of the wild-type level of LH. These findings suggest that the C-terminal extensions are required for wild-type levels of LH.
