Structure and function analysis of enzymes which are

光受容色素の合成に関与する 酵素群の構造機能解析 Structure and function analysis of enzymes which are relative to synthesis of light-reception pigment 2 B 04 竹下恭史朗 1

Introduction • Cyanobacteria have characteristic pigment, which is different from general plants have(chlorophyll etc. ) Thermosynechococcus elongatus BP-1 http: //photosynthesis. c. u-tokyo. ac. jp/SE 00020@. jpg Phycobilin group Pigments of plants, etc. have various absorbance spectra. 2

Introduction • Photosynthesis produces carbohydrates →Phycobilisome as a light-harvesting antenna light carbon dioxide Cell membrane phycobilisome carbohydrates Calvin cycle Photosynthesis electron transport chain cytoplasm Thylakoid membrane Thylakoid lumen Model of cyanobacterial cell 3

Introduction • Phycocyanobilin is synthesized from heme via biliverdin ヘムオキシゲナーゼ ヘム ビリベルジン 4

Introduction • Phycocyanobilin: ferredoxin oxidoreductase(Pcy. A) biliverdin 181, 182 -dihydrobiliverdin Phycocyanobilin Pcy. A Fd 5

Objective : Searching for affinity interaction of Pcy. A and Ferredoxin Method : to research condition(temperature, p. H, etc. ) of complex formation to analyze crystal structure of Pcy. A-Ferredoxin complex Thermosynechococcus elongatus BP-1 http: //photosynthesis. c. u-tokyo. ac. jp/SE 00020@. jpg 6

Result of last year’s research Electrophoresis after overexpression of Pcy. A(27 k. Da) 7

Experiment - amplification of genes • Using outsourced genes(pcy. A, ferredoxin), E. coli DH 5α was transformed and the genes were amplified. • Cell body was spread on LB agar medium and cultivated in 37 ℃ for 16 hours. • Appeared colony was cultivated in LB liquid medium in 16 hours. 8

Experiment - measure of genes • The LB medium was centrifugated and DNA concentration was measured by spectrophotometer. centrifuge extract measure spectrophoto meter precipitation 9

Experiment - digest by restriction enzyme • The genes was processed by restriction enzyme and measured size of the DNA by electrophoresis. TAGGGATCCCTAGG • After verification of DNA size, the genes were done double-digest and cut down the DNA from the gel and do extraction. CATATG GTATAC TAGGGATCCCTAGG 10

Experiment - ligation Gene fragments were processed by using DNA ligase and inserted to p. ET 15 b plasmid. 挿入 得られた遺伝子断片 p. ET 15 b 11

Result and discussion - codon optimization Amino acid Rare codon Usage(%) Amino acid Optimized codon Usage(%) Arg AGA 5 Arg CGC 40 Arg AGG 4 Arg CGC 40 Gly GGA 10 Gly GGC 41 Ile ATA 7 Ile ATT 48 Leu CTA 3 Leu CTG 45 Pro CCC 6 Pro CCG 61 12

Result and discussion - gene concentration Sample name DNA concentration(ng/µl) ferredoxin 1 2. 2 ferredoxin 2 1. 7 ferredoxin 3 26. 4 ferredoxin 4 35. 6 Sample name DNA concentration(ng/µl) pcy. A 1 2. 1 pcy. A 2 86. 6 pcy. A 3 4. 4 pcy. A 4 13. 0 13

Result and discussion - single digest of pcy. A Size of pcy. A and vector = 3, 054 bp Restriction enzyme = Bam. HI ~ATG pcy. A(720 bp) TAGGGATCC~ ~TAC ATCCCTAGG~ Point of pcy. A cut by restriction enzyme Vector=2, 334 bp pcy. A=720 bp Electrophoresis after single-digest 14

Result and discussion - single digest of ferredoxin Size of ferredoxin and vector = 2, 640 bp Restriction enzyme = Bam. HI M ~ATG ferredoxin(306 bp) TAGGGATCC~ ~TAC ATCCCTAGG~ Point of ferredoxin cut by restriction enzyme Vector=2, 334 bp ferredoxin=306 bp Electrophoresis after single-digest 15

Result and discussion - double digest of both genes Size of ferredoxin = 306 bp Size of pcy. A = 720 bp Restriction enzyme = Bam. HI, Nde. I Vector=2, 334 bp Electrophoresis after double-digest ~CATATG fd(306 bp) or pcy. A(720 bp)TAGGGATCC~ ~GTATAC ATCCCTAGG~ Point of ferredoxin and pcy. A cut by restriction enzyme 16

Result and discussion - ligation Sample name DNA concentration(ng/µl) ferredoxin 1 7. 6 ferredoxin 2 7. 2 ferredoxin 3 10. 2 ferredoxin 4 19. 1 Sample name DNA concentration(ng/µl) pcy. A 1 10. 2 pcy. A 2 13. 0 pcy. A 3 23. 2 pcy. A 4 9. 7 17

Conclusion • I got gene fragments which are associated with produce Pcy. A and Ferredoxin. • After this study, I will try expression of the genes and crystalize the proteins. 18