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Foreign Gene Expression in Photosynthetic Bacteria

  • Chapter
The Purple Phototrophic Bacteria

Part of the book series: Advances in Photosynthesis and Respiration ((AIPH,volume 28))

Summary

Large-scale production of membrane proteins in functional form is an arduous task. The overexpression of membrane proteins is fraught with unique challenges, and systems presently in use often fail to generate products which are compatible with subsequent solubilization, stabilization, and purification steps. To address these requirements, it is possible to exploit the unique physiology of the Rhodobacter species of photosynthetic bacteria, which produces extremely large quantities of internal membranes (invaginations of the cytoplasmic membrane) under certain growth conditions in response to changes in light intensity and/or oxygen tension. Towards this end, an expression system has been designed that coordinates synthesis of foreign membrane proteins with synthesis of new membrane into which they can be incorporated. These intracytoplasmic membrane (ICM) vesicles sequestering the newly synthesized foreign proteins are readily isolated by differential centrifugation following cell lysis. A diverse set of foreign membrane proteins — spanning a range of isoelectric points, molecular weights and predicted membrane topologies and representing several prokaryotic and eukaryotic species — has been expressed heterologously in Rhodobacter. Many target membrane proteins can be produced and purified in a semi-automated fashion at levels greater than 10 mg per liter of culture.

This expression system is versatile, offering many strategies to achieve success in expression of membrane proteins. However, it is not necessarily restricted to the production of membrane proteins. For instance, Rhodobacter possesses advantages for the expression of soluble proteins that require an abundance of membrane surface (e.g., proteins only peripherally- or transiently-associated with the membrane) or proteins requiring complex redox cofactors which are native to Rhodobacter. In addition, Rhodobacter ICMs are easily isolated and are naturally enriched in target membrane proteins; thus, these membranes also facilitate biochemical studies, making this expression system valuable for a wide range of applications.

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Abbreviations

DMSO:

dimethyl sulfoxide

E. :

Escherichia

FPLC:

fast protein liquid chromatography

GFP:

green fluorescent protein

HT:

polyhistidine tag

ICM:

intracytoplasmic membrane

IMAC:

immobilized metal affinity chromatography

IPTG:

isopropyl-β-D-thiogalactoside

LH1:

core light-harvesting antenna complex

LH2:

peripheral light-harvesting antenna complex

ORF:

open reading frame

PCR:

polymerase chain reaction

Rba. :

Rhodobacter

RC:

reaction center

SDS:

sodium dodecyl sulfate

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SeMet:

selenomethionine

Tc:

tetracycline

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Authors and Affiliations

  1. Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL, 60439-4803, USA

    Philip D. Laible, Donna L. Mielke & Deborah K. Hanson

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  1. Philip D. Laible
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  3. Deborah K. Hanson
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Correspondence to Philip D. Laible .

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  1. University of Sheffield, UK

    C. Neil Hunter

  2. University of Pennsylvania, USA

    Fevzi Daldal

  3. Argonne National Laboratory, USA

    Marion C. Thurnauer

  4. University of British Columbia, Canada

    J. Thomas Beatty

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© 2009 Springer Science + Business Media B.V

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Laible, P.D., Mielke, D.L., Hanson, D.K. (2009). Foreign Gene Expression in Photosynthetic Bacteria. In: Hunter, C.N., Daldal, F., Thurnauer, M.C., Beatty, J.T. (eds) The Purple Phototrophic Bacteria. Advances in Photosynthesis and Respiration, vol 28. Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-8815-5_42

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