Membrane Protein Expression

in 

Rhodobacter

Background & Significance: 

Membrane proteins are the means by which single-cell organisms interact with their environment and the means by which cells of complex organisms communicate with each other.  Figure 1 shows several typical membrane proteins, including those which simply anchor into the membrane, and those which facilitate transport and communication across cellular membranes.  Not surprisingly, more than half of current pharmaceuticals target transmembrane proteins. Despite their physiological importance, the complexity and relative low abundance of these proteins in tissues (coupled with the difficulty of acquiring human tissue) has been a major hindrance to research efforts.  There are many reviews which address these issues with one recent review being quite comprehensive in its quantitative comparison of protein expression in the dominant expression systems (E.coli, yeasts, and higher eukaryotic cells such as mammalian and insect cells/baculavirus) [Sarramegna et al., 2003]. 



Grant: Grace Woodward Foundation


Abstract: The proposed research seeks to develop Rhodobacter sphaeroides for the production of functional membrane proteins at concentrations approaching grams per liter, which is more an order of magnitude higher than current expression systems. A primary goal is to achieve sufficiently high protein expression to facilitate cost-effective isotopic labeling for NMR characterization of membrane protein structure. Knowing the structure of membrane proteins is helpful for understanding the basis of disease and the design of new drugs to remedy those diseases. This protein expression platform will thereby provide a general route to structural determination of medically important membrane proteins that does not require crystallization for x-ray crystallography. Since the photobioreactor system developed at University Park has provided exceptionally promising preliminary results for the expression of a cytochrome membrane protein in a collaboration with Argonne National Lab, the focus of the proposed research will be to demonstrate breadth of applicability of this expression technology to a series of medical protein targets of increasing complexity that are currently under study at Hershey Medical School. This experimental validation of the approach will set the stage for pursuing funding for both the optimization of the expression system (strain, vector and bioreactor design) as well as expanding the medical targets to more complex membrane protein receptors. Three medical targets related to Alzheimer’s, diabetes, and cancer have been identified, each with an increasing number of transmembrane domains. A screen of expression will initially examine the impact of changing the DNA codons to those used more frequently in Rhodobacter (codon usage optimization). Two different promoters will be tested which are known to drive high level expression of light harvesting proteins in Rhodobacter. Another potential enhancement is a Rhodobacter strain which has altered protein expression which should favorably permit integration of the heterologous membrane proteins into the unique intracellular membrane system of Rhodobacter (which is the basis of the superiority of this expression host over alternative organisms). While examining these different expression candidates and conditions, valuable information on bioreactor control will be determined. The expression approaches determined to provide high concentrations of the membrane protein candidates will be used in the photobioreactor system, along with isotopically labeled media components to produce 15N-labeled membrane proteins. The initial focus will be 15N labeling since it provides the most useful preliminary information at a reasonable cost, while full labeling (15N, 13C, 2H) and NMR analysis requires more extensive funding. These isotopically-labeled proteins will then be used to obtain measurements of atom positions using nuclear magnetic resonance (NMR). The proposed work will provide a ‘proof of concept’ for a general means of membrane protein structural analysis using NMR.

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