The Effect Of PucC Overexpression On Heterologous Protein Expression Levels In Novel Rhodobacter Sphaeroides Expression System

There are many systems used for heterologous protein expression at present,including prokaryotic expression system, eukaryotic expression system et al. However,all of these systems show defects, especially the lack of real-time detection ofrecombinant protein prior to protein purification. It has been reported that Rb.sphaeroides could be employed as a novel host to functionally express membraneprotein.Rhodobacter sphaeroides has been provided an excellent model for studying bothbacterial photosynthesis and membrane development. Under particular growthconditions, a series of events are triggered that cause the differentiation of thecytoplasmic membrane (CM) through a process of invagination into specializeddomains which comprise the intracytoplasmic membrane (ICM). The formation of theICM makes light-harvesting complexes2(LH2), light-harvesting complexes1(LH1)and reaction center (RC) greatly expressed and synthesized. LH2is composed of nineα-subunits and β-subunits, encoded by puc1A and puc1B repectively. Each pair of α/βsubunit binds three Bchl and one Crt in the form of non covalent binding, productingthe functional LH2complex. The functional LH2has maximum absorption at800nmand850nm. The greater formation of LH2leads to the higher spectral absorption.Therefore, Rb. sphaeroides could be developed as a novel system to evaluate theexpression levels of recombinant proteins fused to LH2α-or β-subunit rapidly in realtime prior to extraction using the characteristic spectral properties of host cell culturesbearing the LH2α-or β-subunit fusion protein, However, LH2, LH1, and RC belongs tothe membrane protein, the level of the protein expression is low, so the relatively lowexpression level of heterologous proteins has become the major limitation of this system.So that it could be especially meaningful if a new method was developed to promote theexpression level of heterologous protein.It’s reported that pucC is important to the formation of the LH2. PucC can lead theα and β subunit into the ICM to assemble. The pucC deletion can lead to the LH2misassemble. So in this research, we makes the foreign gene gus and pucB fused, andconstructed an heterologous protein expression vector successfully. We overexpress thegene pucC of the expression vector, and constructed the pucC overexpression vector.With the help of the S17-1, the constructed vector and control guided into the Rhodobacter sphaeroides CQU68. Under the conditions of lower oxygen concetrationsand IPTG, the LH2, LH2β-GUS fusion protein get expressed respectively. Thereal-time PCR of pucC gene, Spectral absorption, SDS-PAGE and Western blottingresults suggested that the level of LH2β-GUS fusion protein in vector that pucC wasoverexpreesed are greater than level of LH2β-GUS fusion protein in which the pucCare not overexpreesed. All of this confirmed that the expression level of β-subunit-GUSfusion protein was significantly up-regulated by pucC overexpression.