Heterologous Expression And Prelimnary Function Analysis Of CsmA In Chloroflexus Aurantiacus

Chloroflexus aurantiacus was the first discovered species belonged to the filamentous anoxygenic phototrophs (FAP) family. It has a unique kind of light-harvesting antenna, chlorosome, which alsofunctions in the Green Sulfur Bacteria. Chlorosomes are the largest photosynthetic antenna system in nature. They are ellipsoid nanoparticles with an interior of specific BChl c aggregates. Solar energy harvested by BChl c is transferred to BChl a in the envelop of chlorosome, and then transferred into photosynthetic reaction center inside the cell membrane.The baseplate is the special part of chlorosome that attached to the cell membrane. Thus, it not only plays a connecting role,but as critical regulation site in energy transfer process as well. In baseplate, the protein CsmA is the most abundant and accounts for around one-half of the total protein content in-the chlorosome.In order to explore the mechanism of energy transfer in this thermophile, it is necessary to study the structure and function of CsmA protein.In this project, we utilized molecular biology and protein chemistry technologiesto study the CsmA protein. The investigationincluded the following experiments:(1) Based on the analysis of published genome of Cfx. aurantiacus in Genbank, specific primers were designed to clone thecsmA gene by PCR. The cloned gene was ligated to pET28a expression vector. The construct was then transformed into the expression host E.coliBL21(DE3).(2)Moreover,directed mutagenesis was designed to investigate the25thamino acid residue(histidine) of CsmA protein.The histidine was mutated to alanine. The csmAH25A was also recombinated to pET28a expression vectors.(3)Inducted with IPTG,the His-tagged protein was expressed in the form of inclusion body. Accordingly, we performed refolding of protein from inclusion body and thrombin cleavage on Ni-NAT column. The optimal refolding conditions for our target protein were found to be at16℃, flow rate at0.5ml/min and mild urea gradient.The efficiency of renaturationwas up to41%. High efficiency of cleavage of thrombin on column was attained under the room temperature after20h reaction.(4)Purified wild-type and mutated proteinsare functionally reconstituted with BChlapurified by HPLC. Absorptionspectra of reconstitution products revealed the25th histidine residue of CsmA is the critical binding site ofBChla. These studies have made the foundation for further research on the structure and function of baseplate of chlorosomes in Cfx. aurantiacus.