Plant Molecular Biology

, Volume 11, Issue 6, pp 821–831 | Cite as

Synthesis of active Olisthodiscus luteus ribulose-1,5-bisphosphate carboxylase in Escherichia coli

  • Scott Newman
  • Rose Ann Cattolico


The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5′ non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus.

Key words

co-transcription enzyme assembly eukaryotic algae heterologous expression Rubisco 


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  1. 1.
    Anderson K, Wilke-Douglas M: Construction and use of a gene bank of Alcaligenes eutrophus in the analysis of ribulose bisphosphate carboxylase genes. J Bact 159: 973–978 (1984).Google Scholar
  2. 2.
    Birnboim HC, Doly J: A rapid alkaline extraction method for screening recombinant plasmid DNA. Nucleic Acids Res 7; 1513–1517 (1979).Google Scholar
  3. 3.
    Burnette WN: “Western blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112: 195–203 (1981).Google Scholar
  4. 4.
    Cannon S, Wang P, Roy H: Inhibition of ribulose bisphosphate carboxylase assembly by antibody to a binding protein. J Cell Biol 103: 1327–1335 (1986).Google Scholar
  5. 5.
    Cattolico RA: Chloroplast evolution in algae and land plants. Trends Ecol Evol 1: 64–67 (1986).Google Scholar
  6. 6.
    Chirgwin JM, Przybyla AE, Macdonald RJ, Rutter WJ: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294–5299 (1979).Google Scholar
  7. 7.
    Christeller JT, Terzaghi BE, Hill DF, Laing WA: Activity expressed from cloned Anacystis nidulans large and small subunit ribulose bisphosphate carboxylase genes. Plant Mol Biol 5: 257–263 (1985).Google Scholar
  8. 8.
    Delaney TP, Cattolico RA: Chloroplast ribosomal DNA organization of the chromophytic alga Olisthodiscus luteus. Plant Mol Biol (in press).Google Scholar
  9. 9.
    Gatenby AA: The properties of the large subunit of maize ribulose bisphosphate carboxylase/oxygenase synthesized in Escherichia coli. Eur J Biochem 144: 361–366 (1984).Google Scholar
  10. 10.
    Gatenby AA, van derVies SM, Bradley D: Assembly in E. coli of a functional multi-subunit ribulose bisphosphate carboxylase from a blue-green alga. Nature 314: 617–620 (1985).Google Scholar
  11. 11.
    Gatenby AA, van derVies SM, Rothstein SJ: Co-expression of both the maize large and wheat small subunit genes of ribulose-bisphosphate carboxylase in Escherichia coli. Eur J Biochem 168: 227–231 (1987).Google Scholar
  12. 12.
    Gibson JL, Tabita FR: Organization of phosphoribulosekinase and ribulose bisphosphate carboxylase/oxygenase genes in Rhodopseudomonas (Rhodobacter) sphaeroides. J Bact 169: 3685–3690 (1987).Google Scholar
  13. 13.
    Goldschmidt-Clermont M: The two genes for the small subunit of RuBP carboxylase/oxygenase are closely linked in Chlamydomonas reinhardtii. Plant Mol Biol 6: 13–21 (1986).Google Scholar
  14. 14.
    Gurevitz M, Somerville C, McIntosh L: Pathway of assembly of ribulosebisphosphate carboxylase/oxygenase from Anabaena 7120 expressed in Escherichia coli. Proc Natl Acad Sci USA 82: 6546–6550 (1985).Google Scholar
  15. 15.
    Hemmingsen SM, Ellis RJ: Purification and properties of ribulosebisphosphate carboxylase large subunit binding protein. Plant Physiol 80: 269–276 (1986).Google Scholar
  16. 16.
    Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).Google Scholar
  17. 17.
    Lemieux C, Turmel M, Seligy VL, Lee RW: The large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase is encoded in the inverted repeat sequence of the Chlamydomonas reinhardtii chloroplast genome. Curr Genet 9: 139–145 (1985).Google Scholar
  18. 18.
    Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).Google Scholar
  19. 19.
    McIntosh L, Poulsen C, Bogorad L: Chloroplast gene sequence for the large subunit of ribulose bisphosphate carboxylase of maize. Nature 288: 556–560 (1980).Google Scholar
  20. 20.
    Newman SM, Cattolico RA: Structural, functional and evolutionary analysis of ribulose-1,5-bisphosphate carboxylase from the chromophytic alga Olisthodiscus luteus. Plant Physiol 84: 483–490 (1987).Google Scholar
  21. 21.
    Newman SM, Cattolico RA: Structural and functional relatedness of chromophytic and rhodophytic Rubisco. In: Biggins J (ed) Progress in Photosynthetic Research, Vol. 4, Kluwer Academic Publishers, Dordrecht, pp. 671–674 (1987).Google Scholar
  22. 22.
    Nierswicki-Bauer SA, Curtis SE, Haselkorn R: Cotranscription of the genes encoding the small and large subunits of ribulose-1,5-bisphosphate carboxylase in the cyanobacterium Anabaena 7120. Proc Natl Acad Sci USA 81: 5961–5965 (1985).Google Scholar
  23. 23.
    Palmer JD, Nugent JM, Herbon LA: Unusual structure of geranium chloroplast DNA: A triple-sized inverted repeat, extensive gene duplications, multiple inversions, and two repeat families. Proc Natl Acad Sci USA 84: 769–773 (1987).Google Scholar
  24. 24.
    Plumley FG, Kirchman DL, Hodson RE, Schmidt GW: Ribulose bisphosphate carboxylase from three chlorophyll c-containing algae. Plant Physiol 80: 685–691 (1986).Google Scholar
  25. 25.
    Reith ME, Cattolico RA: In vivo chloroplast protein synthesis by the chromophytic alga Olisthodiscus luteus. Biochemistry 24: 2556–2561 (1985).Google Scholar
  26. 26.
    Reith ME, Cattolico RA: Inverted repeat of Olisthodiscus luteus chloroplast DNA contains genes for both subunits of ribulose-1,5-bisphosphate carboxylase and the 32000-dalton QB protein: phylogenetic implications. Proc Natl Acad Sci USA 83: 8599–8603 (1986).Google Scholar
  27. 27.
    Sharma SK: On the recovery of genetically engineered proteins from Escherichia coli. Separ Sci Technol 21: 701–726 (1986).Google Scholar
  28. 28.
    Shinozaki K, Sugiura M: The gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is located close to the gene for the large subunit in the cyanobacterium Anacystis nidulans 6301. Nucl Acids Res 11: 6957–6964 (1983).Google Scholar
  29. 29.
    Somerville C, McIntosh L, Fitchen J, Gurevitz M: The cloning and expression in E. coli of RuBP carboxylase/oxygenase large subunit genes. Meth Enzymol 118: 419–433 (1985).Google Scholar
  30. 30.
    Starnes SM, Lambert DH, Maxwell ES, StevensJr. SE, Porter RD, Shively JM: Cotranscription of the large and small subunit genes of ribulose-1,5-bisphosphate carboxylase/oxygenase in Cyanophora paradoxa. FEBS Microbiol Lett 28: 165–169 (1985).Google Scholar
  31. 31.
    Steinmuller K, Kaling M, Zetsch K: In vitro synthesis of phycobiliproteins and ribulose-1,5-bisphosphate carboxylase by non-polyadenylated-RNA of Cyanidium caldarium and Porphyridium aerugineum. Planta 159: 308–313 (1983).Google Scholar
  32. 32.
    Tabita FR, Small CL: Expression and assembly of active cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase in Escherichia coli containing stoichiometric amounts of large and small subunits. Proc Natl Acad Sci USA 82: 6100–6103 (1985).Google Scholar
  33. 33.
    Van denBroeck G, Timko MP, Kausch AP, Cashmore AR, VanMontagu M, Herrera-Estrella L: Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose-1,5-bisphosphate carboxylase. Nature 313: 358–363 (1985).Google Scholar
  34. 34.
    Young RA, Davis RW: Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci USA 80: 1194–1198 (1983).Google Scholar

Copyright information

© Kluwer Academic Publishers 1988

Authors and Affiliations

  • Scott Newman
    • 1
  • Rose Ann Cattolico
    • 1
  1. 1.Department of Botany KB-15University of WashingtonSeattleUSA
  2. 2.Zoology DepartmentDuke UniversityDurhamUSA

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