Heterologous Expression.Purification And Crystallization Of Chlorosome Envelope Proteins From Chloroflexus Aurantiacus

Chloroflexus aurantiacus was the first discovered species belongs to the filamentous anoxygenic phototrophs(FAP)family of photosynthetic bacteria.The light harvesting antenna of FAP called chlorosome,which is the largest photosynthetic antenna system in nature.The Green S?Lfur Bacteria also contain the chlorosome.The gigantic chlorosome encapspuLated by a lipid monolayer is ellipsoid nanoparticle with an interior of self-aggregated BChl c/d/e.Solar energy harvested by BChls is transferred to BChl a containing proteins of the chlorosome envelope,and then transferred into photosynthetic reaction center inside the cell membrane.It was found early that some pigment-protein complexes contained in the chlorosome envelope,in which the protein CsmA,CsmM and CsmN are the most abundant.When the genome of Cfx.aurantiacus was sequenced,it showed that other proteins such as CsmP,CsmO,CsmY were also exist in the chlorosome envelope.In order to explore the photosynthesis mechanism of this thermophile,it is necessary to study the structure of these energy transfer proteins.In this project,we have put into use molecpLar biology and protein chemistry technology to study the chlorosome envelope proteins and executed the following experiments:(1)According to the genome data of Cfx.aurantiacus in NCBI Genbank,on the one hand,specific primers were designed to clone the csmM,csmN,csmO and csmP gene by PCR;on the other hand,joint primers were designed to clone the csmM-N-P-O,csmN-P-O,csmN-P and csmP-O fused sequence by PCR.The cloned gene sequences were ligated to pEASY-Blunt El or pEASY-E2 expression vector and constructed the recombinant plasmids of pEASY-Blunt El-csmM,pEASY-Blunt El-csmN,pEASY-Blunt E1-csmP,pEASY1-E2-csmM-N-P-O,pEASY-Blunt E1-csmN-P-O,pEASY-Blunt E1-csmN-P and pEASY-Blunt E1-csmP-O.The recombinant plasmids were then transformed into the expression host E.coli BL21(DE3).(2)The bioinformatics analysis were carried out for these envelope proteins and the expression status of inducing proteins.(3)After screening the appropriate conditions,the His-tagged proteins were induced expression by addition of IPTG.Afterwards,the target proteins were isolated and purified by using affinity chromatography,ion exchange and gel filtration.(4)Crystallizations of CsmP protein and CsmN-P fusion protein were carried out by the hanging-drop vapor-diffusion method.These experiments lay the theoretical foundation for the extensive study on the structure and function of chlorosome in photosynthetic bacteria.