a strategy to produce membrane proteins for structural genomics
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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 proteins for structural genomics.

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a strategy to produce membrane proteins for structural genomics

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.

additional features of the rhodobacter membrane protein expression system
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

current rhodobacter expression system

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

progress with mcsg membrane protein targets
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

culture and purification
Culture and Purification

Only 1 major step (*) novel to membrane protein purification

Laible et al., 2002

examples of successful expression and purification from first round of targets
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

planned improvements for rhodobacter expression system

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

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