Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum

Gregory S. Orf, Kevin E. Redding

Research output: Contribution to journalArticle

Abstract

The heliobacterial photochemical reaction center (HbRC) from the chlorophototrophic Firmicutes bacterium Heliobacterium modesticaldum is the only homodimeric type I RC whose structure is known. Using genetic techniques recently established in our lab, we have developed a rapid heterologous expression system for the HbRC core polypeptide PshA. Our system relies on rescue of the non-chlorophototrophic ∆pshA::cbp2p-aph3 strain of Hbt. modesticaldum by expression of a heterologous pshA gene from a replicating shuttle vector. In addition, we constructed two tagged variants of PshA, one with an N-terminal octahistidine tag and one with an internal hexahistidine tag, which facilitate rapid purification of pure, active HbRC cores in milligram quantities. We constructed a suite of shuttle vectors bearing untagged or tagged versions of pshA driven by various promoters. Surprisingly, we found that the eno and gapDH_2 promoters from Clostridium thermocellum drive better expression of pshA than fragments of DNA derived from the region upstream of the pshA locus on the Hbt. modesticaldum genome. This “pshA rescue” strategy also provided a useful window into how Hbt. modesticaldum regulates pigment synthesis and growth rate when chlorophototrophic output decreases.

Original languageEnglish (US)
Pages (from-to)335-348
Number of pages14
JournalPhotosynthesis research
Volume142
Issue number3
DOIs
StatePublished - Dec 1 2019

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Keywords

  • Epitope-tagging
  • Heliobacteria
  • Membrane protein
  • Mutagenesis
  • Reaction center

ASJC Scopus subject areas

  • Biochemistry
  • Plant Science
  • Cell Biology

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