BioBrick™ compatible vector system for protein expression in Rhodobacter sphaeroides

Ilya B. Tikh, Mark Held, Claudia Schmidt-Dannert

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

We report here the creation of a modular, plasmid-based protein expression system utilizing elements of the native Rhodobacter puf promoter in a BioBrick™-based vector system with DsRed encoding a red fluorescent reporter protein. A suite of truncations of the puf promoter were made to assess the influence of different portions of this promoter on expression of heterologous proteins. The 3′ end of puf was found to be particularly important for increasing expression, with transformants accumulating significant quantities of DsRed under both aerobic and anaerobic growth conditions. Expression levels of this reporter protein in Rhodobacter sphaeroides were comparable to those achieved in Escherichia coli using the strong, constitutive P lac promoter, thus demonstrating the robustness of the engineered system. Furthermore, we demonstrate the ability to tune the designed expression system by modulating cellular DsRed levels based upon the promoter segment utilized and oxygenation conditions. Last, we show that the new expression system is able to drive expression of a membrane protein, proteorhodopsin, and that membrane purifications from R. sphaeroides yielded significant quantities of proteorhodopsin. This toolset lays the groundwork for the engineering of multi-step pathways, including recalcitrant membrane proteins, in R. sphaeroides.

Original languageEnglish (US)
Pages (from-to)3111-3119
Number of pages9
JournalApplied Microbiology and Biotechnology
Volume98
Issue number7
DOIs
StatePublished - Apr 2014

Keywords

  • BioBricks™
  • Proteorhodopsin
  • Recombinant membrane protein expression
  • Rhodobacter sphaeroides
  • Synthetic biology
  • puf promoter

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