Construction And Applications Of A Novel Expression System For Heterologous Protein Production With Rhodobacter Sphaeroides

Currently, E. coli and yeast have been proven to be the extremely useful heterologous expression systems and are widely used for producing recombinant proteins exhibiting various important biological functions. However, expression levels of the recombinant protein in all of the commercial expression systems can not be evaluated rapidly in real time prior to extraction.Rhodobacter sphaeroides, with completely sequenced genomes, has been provided an excellent model for studying both bacterial photosynthesis and membrane development. Under optimal growth conditions, a series of events are triggered that cause the differentiation of the cytoplasmic membrane (CM) through a process of invagination into specialized domains which comprise the intracytoplasmic membrane (ICM). Meantime, the light-harvesting complexes 2 (LH2), LH1 and reaction center (RC) are greatly synthesized, which could make up over 50% of total membrane protein content. LH2 is composed of nine?-subunits and?-subunits, encoded by puc1A and puc1B of puc1BAC operon, repectively. It has been reported that there is a second puc operon puc2BA involved in the formation of LH2 in Rb. sphaeroides genome. LH2 has maximum absorption at ~800 nm and ~850 nm. The greater formation of LH2 leads to the higher spectral absorption. Therefore, Rb. sphaeroides could be developed as a novel system to evaluate the expression levels of recombinant proteins fused to LH2?- or?-subunit rapidly in real time prior to extraction using the characteristic spectral properties of host cell cultures bearing the LH2?- or?-subunit fusion protein.In the present study, we constructed the puc1BA and puc2BA mutant strain Rb. sphaerodies CQU68 (genomic deletion of puc1BA, puc2BA and pufBALMX) based on DD13 mutant strain (genomic deletion of puc1BA and pufBALMX, lack LH2, LH1 and RC) and constructed the expression vector pRKlacIqpucPpuc1BHis101AC which was suitable for the expression of fusion protein in Rb. sphaeroides. The expression vector contained a hybrid promoter lacIq-pucP-lacO consisting of E. coli lactose operon gene lacIq, lacO and Rb. sphaeroides puc1BAC operon promoter pucP. We further established the experimental methods for one-step rapid purification of functional LH2 and LH2?-subunit from Rb. sphaeroides through affinity chromatography by His-tag. Based on the above experimental results, we finally expressed LH2?-subunit-GFP and LH2?-subunit-HNP3 fusion proteins in CQU68 mutant strain, respectively. Spectral absorption, SDS-PAGE and Western blotting results suggested that the LH2 typical spectral absorption at ~800 nm and ~850 nm could be used as a monitor to evaluate the heterologous protein expression levels.The main experimental results are as follows:1. The constructed powerful hybrid lacIq-pucP-lacO possessed the ability to tightly control the formation of LH2 and LH2?-subunit fusion protein that could be formed only under low oxygen tension (semi-aerobic) with the addition of IPTG. The optimal IPTG concentration for induction was 1 mM.2. LH2 and LH2?-subunit fusion protein with great amount of BChl and Crt, exhibiting high activity in electron transport, was one-step rapidly purified from Rb. sphaeroides through the constructed His-tag, which greatly simplified the LH2 and LH2?-subunit fusion protein purification process compared to the the traditional experimental methods sucrose indensity gradient centrigugation.3. LH2?-subunit-GFP and LH2?-subunit-HNP3 fusion proteins were successfully expressed in Rb. sphaeroides CQU68 mutant strain and were integrated into ICM with the small native membrane LH2?-subunit. The LH2?-subunit fusion protein still had the spectral absorption at ~800 nm and ~850 nm. Spectral absorption analysis, SDS-PGAE and Western blotting results demonstrated that the recombinant protein expression levels turned out to be higher as the host strain cell cultures typical spectral peak heights increase. Consequently, the typical spectral absorption at ~800 nm and ~850 nm could be used as a monitor to rapidly evaluate fusion protein expression levels during cell culture in real time.In the present study, we successfully constructed a novel Rb. sphaeroides expression system that could be used to tightly regulate the expression of heterologous proteins and to rapidly evaluate these proteins expression levels in real time. Thus, the construction of the novel system holds tremendous potential in the study of heterologous protein expression and function.