A Novel Rhodobacter sphaeroides Expression System for Real-Time Evaluation of Heterologous Protein Expression Levels

Zhiping      Zhao; Zongli      Hu; Xin      Nie; Lijing      Cheng; Guolin      Ding; Min      Luo; Yu      Pan; Yan      Liang; Guoping      Chen

Abstract

Heterologous protein expression levels can not be evaluated in real time by experimental procedures commonly used for most expression systems during host cell culture. Rb. sphaeroides has provided an ideal system for studying both photosynthesis and membrane development and exhibited potential as a novel expression system. We constructed the puc1BA and puc2BA mutant strain Rb. sphaeroides CQU68 and used it as a novel expression system to heterologously express proteins fused to β-subunit of light-harvesting 2 complexes (LH2). The presence of LH2 with β-subunit fusion proteins was spectrally detected by the LH2 typical absorption at ∼800 nm and ∼850 nm, and the formation of these complexes were further confirmed by SDS-PAGE and Western blot analysis. The expression levels of heterologous protein measured by SDS-PAGE and Western blot turned out to be higher as the typical spectral peak heights increase. These findings suggested that the production of the heterologous protein could be rapidly detected through the LH2 absorption at ∼800 nm and ∼850 nm. Moreover, the typical absorption could be used as a monitor for rapid and real-time evaluation of heterologous protein expression levels.

Keywords: Expression system, LH2 β-subunit, protein expression levels, Rb. Sphaeroides, real-time, spectral absorptionExpression system, LH2 β-subunit, protein expression levels, Rb. Sphaeroides, real-time, spectral absorption