A powerful hybrid puc operon promoter tightly regulated by both IPTG and low oxygen level.
Rhodobacter sphaeroides has been intensively studied and provides an excellent model for studying both photosynthesis and membrane development. The photosynthetic apparatus (LH2 and LH1-RC complexes) can be synthesized in large scale and integrated into the intracytoplasmic membrane system under specific conditions, which thus provides us insight to utilize the puc or(and) puf operon to heterologously express recombinant proteins in the intracytoplasmic membrane using Rb. sphaeroides as a novel expression system. However, basal level of expression of puc and puf promoter is uncontrolled. We report the construction of LH2 polypeptide expression vector that contains a reengineered lacI(q)-puc promoter-lac operator hybrid promoter, which allows the puc operon to be regulated by both IPTG and low oxygen level. Synthesis of LH2 complexes was completely repressed in the absence of isopropyl beta-D-thiogalactoside (IPTG), and the degree of induction was controlled by varying the concentration of IPTG. The optimal concentration of IPTG was determined. SDS-PAGE and Western blot were employed for further analysis. Our results suggest that the reengineered hybrid promoter is efficient to tightly regulate the expression of the puc operon, and our strategy can open up a new approach in the study of the membrane protein expression system.
Hu
Zongli
Z
Key Laboratory of Biorheological Science and Technology and Bioengineering College, Chongqing University, Chongqing, 400044, PR China. huzongli@cqu.edu.cn
Zhao
Zhiping
Z
Pan
Yu
Y
Tu
Yun
Y
Chen
Guoping
G
D001426:Bacterial Proteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology*; D045342:Light-Harvesting Protein Complexes / Q000378:metabolism; D009876:Operon; D010100:Oxygen / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012242:Rhodobacter sphaeroides / Q000235:genetics* / Q000378:metabolism
C081521:B800-850 light-harvesting complex, bacteria; D001426:Bacterial Proteins; D045342:Light-Harvesting Protein Complexes; D007544:Isopropyl Thiogalactoside; D010100:Oxygen