1 Synthesis of Uniform Polymers with Recombinant DNA Technique Kazuhisa Yano p / = 1 + p 2 M w ( / = 1 ) 1956 Szwarc / = 1.03 M w M w M w M n M n M n M n M w M n DNA
2 1955 Natta AlEt 3 -TiCl 4 Ziegler TiCl 4 TiCl 3 Ziegler- NattaZiegler-Natta Natta TiCl 3 AlEt 2 Cl (PP) 90 1975 Montedison MgCl 2 Ti Classification of polymerization. Difference of molecular weight distribution between a conventional polymer and a living polymer.
3 Kaminsky AlMe 3 (Al(Me)O) 10 20 TiCl 4 Ti(OEt) 4 CpTiCl 3 Ti 100 ( 267 ) ( 225 ) β Stereoregularity of polymer. 100 1869 F. Miescher ( (G) (A) (C) (T) ) 1929 DNA RNA 1928 F. Griffith S S R S O. T. Avery 10 1944DNA DNA 1953 J. D. Watson F. H. C. Crick DNA DNA 1960 DNA DNA DNA restriction endonuclease 1970 Smith Haemophilus influenzae d DNA
4 Structure of DNA. Watson-Crick model of DNA. Boyer EcoR I Roberts 30 100 DNA 1970 Mandel Higa DNA λ 2Cohen R6-5 DNA DNA 1972 ClewellE1 ColE1 2000 3000
5 DNA ColE1 Cohen psc101 DNA RSF2124 ColE1 ( 7.8MDal ) 1977 Bolivar R7268 Cleavage with restriction endonucleases. RSF2124 psc101 ColE1 pmb1 pbr322 1973Cohen Boyer Boyer EcoR I R6-5 Cohen psc101 2 DNA 1 DNA 2 DNA DNA C600 psc101 R6-5 Staphylococcus aureus p1258 Structure of pbr322. psc101 1 Xenopus laevis RNA psc101 DNA 1973
6 mrna DNA α β Cloning of tetracycline resistent plasmid.
7 α β 30 3 (GlyProHyp) Gly ProHyp 20 (GlyProPro) Salerno GGT CCG 2 ( 18 ) DNA 32 DNA Principle of the synthesis of uniform polymers. Potential problems in biosynthesis.
8 9 β Bombyx mori ( GlyAlaGlyAlaGlySer ) Ferrari 6 DNA 160 DNA DNA Cappello ( GlyAlaAlaValThrGlyArgGlyAspSerProAla- SerAlaAlaGlyTyr ) 6 1 ( ValProGlyValGly ) Waite Structure of protein.
9 Salerno Mytilus edulis 10 AlaLysProSerTyrHypHypThrDopaLys Dopa = 3,4-dihydroxy phenylalanine; Hyp = Hydroxy proline Dopa Hyp Phe Pro 30 120 600 600 2 λpl E. coli K12 IG110 T7 E. coli BL21 40 60 DNA α β DeGrado C 4 α ( GlyGluLeuGluGluLeuLeuLysLysLeuLysGluLeu- LeuLysGly ) E. coli Lawton ( AsnLeuAlaPheValAlaGlnTrp ) α 36bp 576bp DNA A Tirrell GlyAla β β β (AlaGly) 3 ProGluGly E. coli 100mg / 10L (AlaGly) β ProGluGly β (AlaGly) 4 - ProGluGly X β 30A Structure of four helix bundle. Monomer DNA sequence of [ (AlaGly) 3 ProGluGly ] n.
10 β β (AlaGly) 3 GluGly X β ProGluGly GluGly (AlaGly) Strategy for cloning and expression of [ (AlaGly) 3 ProGluGly ] n.
11 Expected structure of [ (AlaGly) 3 ProGluGly ] n γ- α L- PBLG α γ- α L- HGluAsp(Glu 17 Asp) 4 GluGluOH E. coli / 1.2 1.38 α L- M w M n PLGA) 20 (SeMet) (GlyAla) 3 GlySeMet SeMet SeMet 100 Comparison of molecular weight distribution of HGluAsp(Glu 17 Asp) 4 - GluGluOH with two commercial PLGAs on a 12% polyacrylamide gel. Lane 1, PLGA of molecular weight 9050 and polydispersity Index 1.20; Lane 2, HGluAsp(Glu 17 Asp) 4 GluGluOH; Lane 3, PLGA of molecular weight 21,000 and polydispersity Index 1.38. 1) P. J. Flory (1980) 304 2) Szwarc, M., et al. : J. Am. Chem. Soc., (1956), 2656 3) Miyamoto, M., et al. : Macromolecules, (1984), 265 4) Faust, R. and Kennedy, J. P. : Polym. Bull., (1986), 317
12 5) Doi, Y., et al. : Macromolecules, (1979), 814 6) Yasuda, H., et al. : J. Am. Chem. Soc., (1992), 4908 7) Natta, G. : Atti Accad. Naz. Lincei Cl. Sci. Fis. Mat. Nat. Rend., -4(1955), 61 8) 7, (1969), 261p., 9) 3 56-39767 10), (1985), 59 11) Sinn, H. and Kaminsky, W. : Adv. Organomet. Chem., (1980), 99 12) Ishihara, N., et al. : Macromolecules, (1986), 2464 13), (1969), 402p. 14) Chatani, Y. : Prog. Polym. Sci. Jpn., (1974), 149 15) No. 40, (1989), 18, 16) Panayatov, I. M. and Rashkov, I. B. : Makromol. Chem., (1974), 3305 17) Tieke, B. and Wegner, G. : Makromol. Chem. Rapid Commun., (1981), 543 18) Inoue, H. and Yoneyama, H. : J. Electroanal. Chem., (1987), 291 19), (1987), 157p., 20) Smith, H. O. and Wilcox, K. W. : J. Mol. Biol., (1970), 379 21) Kelly, T. J., Jr. and Smith, H. O. : J. Mol. Biol., (1970), 393 22) Yoshimori, R. N. : A Genetic and Biochemical Analysis of the Restriction and Modification of DNA by Resistance Transfer Factors, (1971), Ph. D. Thesis, Univ. of California, San Francisco Medical Center 23) Roberts, R. J. : Gene, (1978), 183 24) Mandel, M. and Higa, A. : J. Mol. Biol., (1970), 159 25) Cohen, S. N., et al. : Proc. Natl. Acad. Sci. USA, (1972), 2110 26) Clewell, D. B. : J. Bacteriol., (1972), 667 27) Old, R. W. and Primrose, S. B. ( (1991), 55 28) Bolivar, F. R., et al. : Gene, (1977), 75 29) Cohen, S. N., et al. : Proc. Natl. Acad. Sci. USA, (1973), 3240 30) Chang, A. C. Y. and Cohen, S. N. : Proc. Natl. Acad. Sci. USA, (1974), 1030 31) Morrow, J. F., et al, : Proc. Natl. Acad. Sci. USA, (1974), 1743 32), (1982), 78, 33) Chou, P. Y. and Fasman, G. D. : Adv. Enzymol., (1978), 45 34) (1988), 215, 35) Goldberg, I., et al. : Gene, -2(1989), 305 36) Goldberg, I. and Salerno, A. J. : Mater. Reseach. Soc. Symp. Proc., (1990), 229 37) Brutlag, D., et al. : Cell, (1977), 509 38) Hartley, J. L. and Gregori, T. J. : Gene, (1981), 347 39) Lucas, F., et al. : Biochem. J., (1957), 468 40) Ferrari, F. A., et al. : WO 88-03533 41) Cappello, J. : MRS Bull., -10(1992), 48 42) Sandberg, L. B., et al. : New Engl. J. Med., (1981), 566 43) Cappello, J., et al. : Biotechnol. Prog., (1990), 198 44) Waite, J. H. : Biochemistry, (1985), 5010 45) Laursen, R. A., et al. : Mater. Research Soc. Symp. Proc., (1990), 237 46) Salerno, A. J. and Goldberg, I. : Polym. Prepr. ( Am. Chem. Soc., Div. Polym. Mater. ), (1992), 398 47) Eisenberg, D., et al. : Proteins Struct. Funct. Genet., (1986), 16 48) Chothia, C. : Annu. Rev. Biochem., (1984), 537 49) DeGrado, W. F., et al. : Cold Spring Harbor Symp. Quant. Biol., (1987), 521 50) Lawton, C. W., et al. : Mat. Res. Soc. Symp. Proc., (1990), 251 51) Fraser, R. D. B., et al. : J. Mol. Biol., (1965), 706 52) Chou, P. Y. and Fasman, G. D. : J. Mol. Biol., (1977), 135 53) Chou, P. Y. and Fasman, G. D. : Biochemistry, (1974), 211 54) McGrath, K. P., et al. : J. Am. Chem. Soc., -2(1992), 727 55) Creel, H. S., et al. : Macromolecules, (1991), 1213 56) Krejchi, M. T., et al. : Polym. Prepr., -1(1991), 411 57) Zhang, G., et al. : Macromolecules, -13(1992), 3601 58) Dougherty, M. J., et al. : Macromolecules, (1993), 1779 59), (1979), 137 60) 3 (1990) 67 61) ref. 60), p.68 62) (1983), 5 63) Tirrell, D. A., et al. : MRS Bull., -7(1991), 24 64) ref. 60), p.13 14 1958