TY - JOUR
T1 - BioBrick™ compatible vector system for protein expression in Rhodobacter sphaeroides
AU - Tikh, Ilya B.
AU - Held, Mark
AU - Schmidt-Dannert, Claudia
PY - 2014/4
Y1 - 2014/4
N2 - 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.
AB - 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.
KW - BioBricks™
KW - Proteorhodopsin
KW - Recombinant membrane protein expression
KW - Rhodobacter sphaeroides
KW - Synthetic biology
KW - puf promoter
UR - http://www.scopus.com/inward/record.url?scp=84898919229&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84898919229&partnerID=8YFLogxK
U2 - 10.1007/s00253-014-5527-8
DO - 10.1007/s00253-014-5527-8
M3 - Article
C2 - 24509770
AN - SCOPUS:84898919229
VL - 98
SP - 3111
EP - 3119
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
SN - 0175-7598
IS - 7
ER -