Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum
| Autor: | Kevin Redding, Gregory S. Orf |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
0301 basic medicine Photosynthetic Reaction Center Complex Proteins Heterologous Plant Science Photochemistry 01 natural sciences Biochemistry 03 medical and health sciences chemistry.chemical_compound Bacterial Proteins Shuttle vector Histidine Promoter Regions Genetic Gene Clostridiales biology Chemistry Promoter Pigments Biological Cell Biology General Medicine Photochemical Processes biology.organism_classification Recombinant Proteins 030104 developmental biology Heliobacteria Clostridium thermocellum Heterologous expression Microorganisms Genetically-Modified DNA Molecular Chaperones 010606 plant biology & botany |
| Zdroj: | Photosynthesis Research. 142:335-348 |
| ISSN: | 1573-5079 0166-8595 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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