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(TM)-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.
journal_name
Appl Microbiol Biotechnoljournal_title
Applied microbiology and biotechnologyauthors
Tikh IB,Held M,Schmidt-Dannert Cdoi
10.1007/s00253-014-5527-8subject
Has Abstractpub_date
2014-04-01 00:00:00pages
3111-9issue
7eissn
0175-7598issn
1432-0614journal_volume
98pub_type
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