A Powerful Hybrid puc Operon Promoter Tightly Regulated by Both IPTG and Low Oxygen Level

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Zongli Hu^#, Zhiping Zhao^#, Yu Pan, Yun Tu, and Guoping Chen^*

Key Laboratory of Biorheological Science and Technology and Bioengineering College, Chongqing University, 174 Shapingba Main Street, Chongqing 400044, People’s Republic of China; fax: (0086) 23-6511-2674; E-mail: chenguoping@cqu.edu.cn; huzongli@cqu.edu.cn; zhipingzhao@cqu.edu.cn; ipying@163.com; tuyun@cqu.edu.cn

^# These authors contributed equally to this work.

^* To whom correspondence should be addressed.

Received September 8, 2009; Revision received November 5, 2009

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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 β-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.

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KEY WORDS: LH2, hybrid promoter, IPTG, Rhodobacter sphaeroides

DOI: 10.1134/S0006297910040176

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