Site-directed Mutation Of The Gene Slr2049of Synechocystis Sp．PCC6803and The Study On The Function

In2006, Zhao and his colleagues have found a bilin lyase gene cpeS (named asalr0617) in Anabaena sp. PCC7120which can encode bilin lyase CpeS catalyzephycocyanobilin (PCB) attach to the site cysteine-84of β-subunits of phycocyanin orphycoerythrin. Though homology analyzes by software, we have found gene slr2049inSynechocystis sp. PCC6803is parallel and highly homology to gene cpeS. Ourpreliminary study of gene slr2049confirmed the protein Slr2049has similar functionwith protein CpeS, is a bilin lyase.In order to further study and understanding on the relationship between constructionand function of gene slr2049.Our experiment adapted site-directed mutagenesis to mutatesome amino acid sites of the protein Slr2049. For the selected amino acid sites, we usealignment software to compare the sequence of protein Slr2049and other six proteinswhich have the same function with CpeS, picked eight of gained twenty-two conservativeamino acid sites, not only the picked amino acid sites, but also consider the structure andphysicochemical properties. We need three other related genes in E. coli to synthesisβ-subunit of fluorescence phycocyanin, they are heme oxygenase1gene(ho1),phycocyanin ferredoxin reductase gene(pcyA) and apoprotein β-subunit gene(cpcB).Fristextract the whole genome of Synechocystis sp. PCC6803by the improved approach,then add the relevant cleavage sites in the design of primers,get the target fragment withPCR. We construct wild-type slr2049, eight mutants slr2049and clone vectors of thethree related genes, then get paired together these genes to construct the expression vectoras pCDF-cpcB-slr2049wild-type, eight pCDF-cpcB-slr2049mutants and pET-ho1-pcyA,put wild-type and mutant expression vector into E. coli BL21respectively byco-transformation. By the induction of IPTG, get recombinant protein from heterologousrecombinant in vivo, because the vector carrying the histidine tag, the gainedrecombinant proteins of wild-type and mutants can be purified with nickel affinitychromatograph.Then we get the absorption spectrometric data of proteins respectively.All the recombiant phycobiliproteins from wild-type and mutants were separated viaSDS-PAGE, and their absorption spectra and fluorescence emission spectra wererecorded. Results showed that phycobiliprotein from wild-type had an absorption peak at623nm and a emission extreme at640nm, neither detectable “blue” nor “red” shift wasfound in the absorption or emission curve of phycobilirprotein from mutants slr2049 (H21S), slr2049(L23S), slr2049(F25S), slr2049(W72L), slr2049(G84S), slr2049(Y124S).Mutants slr2049(L23S), slr2049(W72L), and slr2049(G84S) had significantly lowerabsorption, while the peaks on the spectral curve were also corresponding to thephycobiliprotein. No such spectral characteristics were observed for the mutantsslr2049(A24S) and slr2049(R107S). As spectrum differences of phycobiliproteins wererelative to the different catalytic ability of lyase, among these conserved amino acid sitewhich were analyzed above, we concluded that Ala(24) and Arg(107) were essential forphycobilin attachment, maybe the two positions were the activesites of lyase; His(21),Phe(25) and Tyr(124) have no influence on the intact phycobiliprotein assemble; Leu(23),Try(72) and Gly(84) were necessary for the correct folding.To verify whether the recombinant protein and other two mutant proteins haveexpressed essentially, we use Slr2049to get antibody and set control group to do westernblot. Though western blot, the slr2049wild-type and slr2049(H21S)/slr2049(L23S)/slr2049(F25S)/slr2049(W72L)/slr2049(G84S)/slr2049(Y124S) mutants haveno present absence, however, slr2049(A24S)/slr2049(R107S) did not synthesisβ-phycocyanin. We conclude that slr2049(A24S)/slr2049(R107S) have no activity oflyase and these two sites are crucial to bilin lyase synthesis.