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Bacterial expression of a eukaryotic membrane protein in fusion to various Mistic orthologs

Hay Dvir1, Senyon Choe

  • 1Structural Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.

Protein Expression and Purification
|June 16, 2009
PubMed

Insights

Mistic proteins enhance eukaryotic membrane protein expression in bacteria. Higher Mistic membrane association correlates with increased expression of fused membrane proteins like the aKv1.1 channel.

Area of Science:

  • Molecular Biology
  • Protein Expression
  • Membrane Protein Biochemistry

Background:

  • Mistic proteins, found in Bacillus species, are known to enhance eukaryotic membrane protein expression in bacterial systems.
  • While full-length Mistic (M110) from B. subtilis expresses at the E. coli membrane, shorter orthologs exhibit varying degrees of cytoplasmic localization and membrane affinity.
  • This differential localization suggests a potential link between Mistic's membrane association and its efficacy in protein expression.

Purpose of the Study:

  • To investigate the hypothesis that the expression level of membrane proteins fused to Mistic is directly correlated with the Mistic protein's degree of membrane association.
  • To compare the effectiveness of different Mistic variants as fusion partners for enhancing the expression of the Aplysia californica Kv1.1 (aKv1.1) channel.

Main Methods:

  • Various Mistic proteins were utilized as fusion partners with the aKv1.1 channel, a model eukaryotic membrane protein.
  • Expression levels and membrane association of these fusion proteins in E. coli were analyzed.
  • A chimeric Mistic protein (H1-M4) was constructed and tested to assess the role of specific Mistic domains.

Main Results:

  • Mistic from B. atrophaeus (M4), exhibiting high membrane association, significantly enhanced the expression of the aKv1.1 channel's transmembrane domain.
  • Mistic variant M1, a soluble protein, showed minimal capacity to enhance channel expression.
  • Fusion proteins involving M110, M4, and the H1-M4 chimera demonstrated high quantity and quality expression of the aKv1.1 channel, suitable for structural studies.

Conclusions:

  • The expression levels of 'Misticated' membrane proteins are dependent on the Mistic protein's intrinsic membrane association, acting as an independent chaperoning mechanism.
  • This effect is mediated by direct membrane association rather than sequence-specific interactions with the E. coli translocon machinery.
  • Mistic variants with strong membrane affinity are superior for enhancing the expression of challenging membrane proteins like ion channels.

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