Membrane Protein Engineering
Membrane protein expression system
The cell membrane serves as the interface between an organism and its environment, and internal membranes in eukaryotes separate functional compartments within cells. Proteins inserted in these membranes carry out many essential biological processes including uptake of nutrients, excretion of wastes, signal transduction, and response to external stimuli. In addition, membrane proteins are used in elaborate bioenergetic schemes to fuel all normal cellular activities in healthy organisms. In this post-genomic era, about 35% of the genes in any genome encode membrane proteins. The fraction of proteins associated with the membrane in eukaryotes may be even higher (up to 40%). Notably, membrane proteins constitute the majority of drug targets, thus knowledge of the structures of these proteins would contribute greatly to our understanding of biological processes. Unfortunately, structural information for membrane proteins is exceedingly scarce. It is notoriously difficult to purify quantities of native material that are sufficient for crystallization attempts. As a result, to date, the three-dimensional structures of ~60 unique transmembrane proteins are known in comparison to the structures of representatives of more than ~4000 soluble protein families.
While established technology can routinely generate sufficient quantities of soluble protein for structural studies, no general systems exist or are routinely employed which are tailored specifically for the overexpression of membrane proteins. Although prokaryotic systems have many advantages (simplicity, inexpensive, rapid growth, etc.), the overexpression of membrane proteins in these systems most often results in precipitation of the targeted membrane protein as inclusion bodies (insoluble aggregates within cells). These aggregates result because the native membranes of E. coli do not have the capacity to house the extra protein load of the overexpressed target protein, and no new membrane is synthesized when protein synthesis is induced.
In contrast, photosynthetic bacteria are particularly responsive to changes in light intensity and/or oxygen tension and produce extremely large quantities of internal membranes upon manipulation of conditions to mimic a switch of the organism’s growth mode to photosynthetic. This new intracytoplasmic membrane (ICM) is formed by invaginations of the cell membrane and is produced to house the coordinately-induced transmembrane proteins constituting the photosynthetic machinery of the cell. Upon cell disruption, the intracytoplasmic membrane invaginations break apart from the host’s cytoplasmic membrane becoming sealed ‘inside-out’ particles which, by virtue of their size, are easily isolated with differential centrifugation. These vesicles are rich in the integral membrane proteins that constitute the photosynthetic apparatus. Expression of the ICM and expression of its associated proteins, while coordinated, are not obligatorily linked. Mutant strains lacking some or all of the photosynthetic proteins still elaborate the ICM when appropriately induced.
We are exploiting the unique physiology of R. sphaeroides for the heterologous expression of membrane proteins. Under standardized, semi-aerobic growth conditions, synthesis of the photosynthetic apparatus is autoinduced as the cell density increases and the oxygen tension decreases. We have taken advantage of this property to develop an expression system that replaces genes for photosynthetic proteins with genes encoding heterologous membrane proteins. The expression system consists of a host strain carrying deletions of genes encoding proteins of the photosynthetic apparatus and a plasmid vector carrying cloning sites for insertion of foreign genes. Heterologous expression is driven by a promoter that normally drives expression of the deleted genes encoding photosynthetic proteins. With this host/vector combination, genes encoding membrane proteins of interest can be induced by the environmental cues that also induce the ICM, and the overexpressed protein products can be incorporated into and purified from the ICM.
In recent years, several basic issues involved with the construction of the Rhodobacter membrane protein expression system have been addressed. Expression vectors have been designed and successfully constructed that facilitate the shuttling of genes encoding membrane proteins from foreign organisms into R. sphaeroides for expression and purification. These platform vectors utilize either ligation-dependent or ligation-independent cloning strategies and are designed to fuse a C-terminal polyhistidine tag to the target gene such that the foreign gene products can be efficiently purified by affinity chromatography. The platform vectors are based upon a broad-host-range vector, pRK404, whose sequence has been recently determined. Concomitantly, various strains of R. sphaeroides differing in the complement of native proteins present in their ICM have been evaluated as hosts for heterologous expression of membrane proteins. These strains span the range from a true wild-type to an engineered strain that is deleted for three pigment-protein complexes of the photosynthetic apparatus. The R. sphaeroides host lacking all structural genes of the photosynthetic apparatus is currently in use in a semi-automated production mode.
In the initial analysis of a large set of target proteins, overexpression of the foreign proteins in Rhodobacter membranes is observed for ~60% of the expression constructs. Protein production levels in these expression strains range from 0.5 mg purified protein/L culture to > 20 mg purified protein /L culture, levels which exceed those seen for recombinant expression of native proteins of the ICM (~10 mg purified protein/L culture). In addition, methods have recently been developed for the biosynthetic substitution of methionine by selenomethionine (SeMet) in induced membrane proteins overexpressed in R. sphaeroides and R. capsulatus. These results augment the strategy for using Rhodobacter as a bacterial “factory” for the production of heterologous membrane proteins for structure determination.
