A strategy to produce membrane proteins for structural genomics

1. Invaginations of the cell membrane unique to species of Rhodobacter Model of Rhodobacter cells underscoring key features Electron micrographs of two Rhodobacter deletion strains Laible et al., 2002 A strategy to produce membrane proteinsfor structural genomics Advantage of the Rhodobacter expression system: This organism can be engineered to provide coordinated synthesis of foreign membrane proteins with synthesis of new membrane into which they can be incorporated.

2. Additional features of the Rhodobacter membrane protein expression system • Membrane invaginations are easily isolated by ultracentrifugation • Cell color indicates correct induction conditions for expression of the host membrane and foreign genes • Relatively high expression yields of hypothetical membrane proteins of E. coli have been observed • SeMet is readily incorporated into induced proteins with similar yield and purity issues as soluble SeMet protein derived from E. coli Laible et al., 2002

3. Expression vector (traditional ligation methodologies shuttle foreign genes in place of antenna genes in the puf operon) Rhodobacter host (an engineered strain that lacks all antenna and RC proteins of the photosysnthetic apparatus) Current Rhodobacter Expression System Laible et al., 2002

4. Progress with MCSG membrane protein targets 150 membrane proteins are currently being pursued • Target selection • Concentrate on membrane proteins from E. coli that have no known homolog in the PDB • If a Rhodobacter homolog of the E. coli target exists, then it is also aggressively pursued • Maximize information obtained from a single structure by focusing on protein families with many members • Select targets exhibiting a wide range of MW, pIs, and hydropathy plot signatures • Progress • Targets cloned with > 90% efficiency • Expression analysis is underway • Initial screening shows ~15% of clones express well; some have expression levels that rival those of native proteins of the photosynthetic apparatus Laible et al., 2002

5. Culture and Purification Only 1 major step (*) novel to membrane protein purification Laible et al., 2002

6. Examples of successful expression and purification from first round of targets Expression levels of several proteins rival or exceed levels of highly-expressed, native Rhodobacter membrane proteins. Laible et al., 2002

7. Acknowledgments NIH (R01 GM61887) Planned improvements for Rhodobacter expression system • Employ smaller broad-host-range vector • Introduce LIC methodologies • Automate purification • Optimize ribosome binding site • Investigate leader sequences and placement of affinity tag • Co-express chaperones • Knock out proteases Laible et al., 2002