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

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 ΔpshA::cbp2p-aph3 strain of Hbt. modesticaldum by expression of a heterologous pshA gene from a replicating shuttle vector. Furthermore, 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 additionally provided a useful window into how Hbt. modesticaldum regulates pigment synthesis and growth rate when chlorophototrophic output decreases.

Authors:
ORCiD logo [1]; ORCiD logo [1]
  1. Arizona State Univ., Tempe, AZ (United States)
Publication Date:
Research Org.:
Arizona State Univ., Tempe, AZ (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences, and Biosciences Division
OSTI Identifier:
1631822
Grant/Contract Number:  
SC0010575
Resource Type:
Accepted Manuscript
Journal Name:
Photosynthesis Research
Additional Journal Information:
Journal Volume: 142; Journal Issue: 3; Journal ID: ISSN 0166-8595
Publisher:
Springer
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; reaction center; heliobacteria; epitope-tagging; membrane protein, mutagenesis

Citation Formats

Orf, Gregory S., and Redding, Kevin E. Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum. United States: N. p., 2019. Web. https://doi.org/10.1007/s11120-019-00672-3.
Orf, Gregory S., & Redding, Kevin E. Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum. United States. https://doi.org/10.1007/s11120-019-00672-3
Orf, Gregory S., and Redding, Kevin E. Sat . "Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum". United States. https://doi.org/10.1007/s11120-019-00672-3. https://www.osti.gov/servlets/purl/1631822.
@article{osti_1631822,
title = {Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum},
author = {Orf, Gregory S. and Redding, Kevin E.},
abstractNote = {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 ΔpshA::cbp2p-aph3 strain of Hbt. modesticaldum by expression of a heterologous pshA gene from a replicating shuttle vector. Furthermore, 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 additionally provided a useful window into how Hbt. modesticaldum regulates pigment synthesis and growth rate when chlorophototrophic output decreases.},
doi = {10.1007/s11120-019-00672-3},
journal = {Photosynthesis Research},
number = 3,
volume = 142,
place = {United States},
year = {2019},
month = {9}
}

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