Advertisement

Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Cloning and sequencing of the cDNA encoding the major albumin of Theobroma cacao

Identification of the protein as a member of the Kunitz protease inhibitor family

  • 29 Accesses

  • 27 Citations

Abstract

The major albumin, a polypeptide of 21 kilodaltons (kDa), from the seeds of cocoa (Theobroma cacao L.), has been identified and partially purified by preparative gel electrophoresis. Some N-terminal sequence was obtained, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy DNA (cDNA) clone from a library made from poly(A)+ RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 221-amino-acid polypeptide of Mr 24003. The existence of a precursor to the 21-kDa polypeptide of this size was confirmed by immunoprecipitation from total poly(A)+ RNA translation products. The polypeptide has a hydrophobic signal sequence of 26 amino acids before the mature start, and the mature polypeptide would have an Mr of 21223. The protein sequence is homologous with sequences of the Kunitz protease and α-amylase inhibitor family, and the protein probably functions to defend the seed's protein reserves from the digestive enzymes of invading pests. However because the protein comprises 25–30% of the total seed protein it may itself also function as a storage protein. Electron micrographs of immunogold-labelled embryo sections show that the protein is located in membrane-enclosed organelles.

This is a preview of subscription content, log in to check access.

Abbreviations

cDNA:

copy DNA

IgG:

immunoglobulin G

kb:

kilobase pairs

kDa:

kilodaltons

Mr :

relative molecular mass

SDS-PAGE:

sodium dodecyl sulphate-polyacylamide gel electrophoresis

References

  1. Biehl, B., Wewetzer, C., Passern, D. (1982) Vacuolar (storage) proteins of cocoa seeds and their degradation during germination and fermentation. J. Sci. Food Agric. 33, 1291–1304

  2. Cleveland, D.W., Fischer, S.G., Kirschner, M.W., Laemmli, U.K. (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102–1106

  3. Cuming, A.C., Williams, R.S., Cullimore, J.V. (1986) The use of antibodies in molecular biology. In: Immunology in plant science, pp. 137–154, Wang, T.L., ed. Cambridge University Press, Cambridge, UK

  4. Fritz, P.J., Fritz, K.A., Kauffman, J.M., Patterson, G.R., Robertson, C.A., Stoesz, D.A., Wilson, M.R. (1985) Cocoa seeds: changes in protein and polysomal RNA during development. J. Food Sci. 50, 946–950

  5. Garcia-Olmedo, F., Salcedo, G., Sanchez-Monge, R., Gomez, L., Royo, J., Carbonero, P. (1987) Plant proteinaceous inhibitors of proteinases and α-amylases (Oxford surveys of plant molecular and cell biology 4) 275–334

  6. Gubler, U., Hoffman, B.J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25, 263–269

  7. Hall, T.C., Ma, Y., Buchbinder, B.U., Pyrne, J.W., Sun, S.M., Bliss, F.A. (1978) Messenger RNA for GI protein of french bean seeds: cell-free translation and product characterisation. Proc. Natl. Acad. Sci. USA. 75, 3196–3200

  8. Hilder, V.A., Gatehouse, A.M.R., Sheerman, S.E., Barker, R.F., Boulter, D. (1987) A novel mechanism of insect resistance engineered into tobacco. Nature 330, 160–163

  9. Hilder, V.A., Barker, R.F., Samour, R.A., Gatehouse, A.M.R., Gatehouse, J.A., Boulter, D. (1989) Protein and cDNA sequences of Bowman-Birk protease inhibitors from the cowpea (Vigna unguiculata). Plant Mol. Biol. 13, 701–710

  10. Hill, S.A. (1984) Methods in plant virology. Blackwell, Oxford London

  11. Hoffman, L.M., Sengupta-Gopalan, C., Paaren, H.E. (1984) Structure of soybean Kunitz trypsin inhibitor mRNA determined from cDNA using oligonucleotide primers. Plant Mol. Biol. 3, 111–117

  12. Joubert, F.J., Heussen, C., Dowdle, E.B.D., (1985) Complete amino-acid sequence of trypsin inhibitor DE-3 from Erythrina latissima seeds. J. Biol. Chem. 260, 12948–12953

  13. Koide, T., Ikenaka, T. (1973a) Amino-acid sequence around the reactive site of soybean trypsin inhibitor. Eur. J. Biochem. 32, 408–416

  14. Koide, T., Ikenaka, T. (1973b) Amino-acid sequence of the carboxyl-terminal region and the complete amino-acid sequence of the soybean trypsin inhibitor (Kunitz). Eur. J. Biochem. 32, 417–431

  15. Kreil, G. (1981) Transfer of proteins across membranes. Annu. Rev. Biochem. 50, 317–348

  16. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685

  17. Laskowski, M., and Kato, I. (1980) Protein inhibitors of proteinases. Annu. Rev. Biochem. 49, 593–626

  18. Lazaro, A., Sanchez-Monge, R., Salcedo, G., Paz-Ares, J., Carbonero, P., Garcia-Olmedo, F. (1988) A dimeric inhibitor of insect α-amylase from barley. Cloning of the cDNA and identification of the protein. Eur. J. Biochem. 172, 129–134

  19. Lehrian, D.W., Patterson, G.R. (1987) Cocoa fermentation. In: Biotechnology: a comprehensive treatise, Vol. 5, pp. 529–575, Rehn, H.-J., Reed, G., eds. Verlag Chemie, Weinheim, FRG

  20. Maeda, K. (1986) The complete amino-acid sequence of the endogenous α-amylase inhibitor in wheat. Biochim. Biophys. Acta 871, 250–256

  21. Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular cloning: a laboratory manual. Cold Spring Harbour laboratory

  22. Mason, P.J., Williams, J.G. (1985) Hybridisation in the analysis of recombinant DNA. In: Nucleic acid hybridisation: a practical approach, Hames, B.D., Higgins, S.J., eds. IRL Press, Oxford Washington

  23. Mundy, J., Svendsen, I., Hejgaard, J. (1983) Barley α-amylase-subtilisin inhibitor. I. isolation and characterisation. Carlsberg Res. Commun. 48, 81–90

  24. Pearson, W.R., Lipman, D.J. (1988) Improved tools for biological sequence analysis. Proc. Natl. Acad. Sci. USA 85, 2444–2448

  25. Proudfoot, N.J., Brownlee, G.G. (1976) 3 non-coding region sequences in eukaryotic messenger RNA. Nature 263, 211–214

  26. Ryan, C.A. (1973) Proteolytic enzymes and their inhibitors in plants. Annu. Rev. Plant Physiol. 24, 173–196

  27. Sanger, F., Nicklen, S., Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467

  28. Shibata, H., Hara, S., Ikenaka, T. (1988) Amino-acid sequence of winged bean (Psophocarpus tetragonolobus) chymotrypsin inhibitor, WCI-3. J. Biochem. (Tokyo) 104, 537–543

  29. Slot, J.W., Geuze, H.J. (1981) Sizing of protein A-colloidal gold probes for immuno electron microscopy. J. Cell Biol. 90, 533–536

  30. Staden, R. (1986) The current status and portability of our sequence handling software. Nucleic Acids Res. 14, 217–231

  31. Svendsen, I., Hejgaard, J., Mundy, J. (1986) Complete amino-acid sequence of the α-amylase/subtilisin inhibitor from barley. Carlsberg. Res. Commun. 51, 43–50

  32. Sweet, R.M., Wright, H.T., Janin, J., Chothia, C.H., Blow, D.M. (1974) Crystal structure of the complex of porcine trypsin with soybean trypsin inhibitor (Kunitz) at 2.6A resolution. Biochemistry 13, 4212–4228

  33. Vehar, G.A., Keyt, B., Eaton, D., Rodriguez, H., O'Brien, D.P., Rotblat, F., Oppermann, H., Keck, R., Wood, W.I., Harkins, R.N., Tuddenham, E.G.D., Lawn, R.M., Capon, D.J. (1984) Structure of human factor VIII. Nature 312, 337–342

  34. Von Heijne, G. (1983) Patterns of amino-acids near signal-sequence cleavage sites. Eur. J. Biochem. 133, 17–21

  35. Woods, D.E., Markham, A.F., Ricker, A.T., Goldberger, G., Colten, H.R. (1982) Isolation of cDNA clones for the human complement protein factor B, a class III major histocompatability complex gene product. Proc. Natl. Acad. Sci. USA 79, 5661–5665

  36. Yamamoto, M., Hara, S., Ikenaka, T. (1983) Amino-acid sequences of two trypsin inhibitors from winged bean seeds (Psophocarpusetragonolobus). J. Biochem. (Tokyo) 94, 849–863

Download references

Author information

Additional information

The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies, and to Dr G. Cope, of the Biological Sciences Electron Microscopy Unit, Sheffield University, for taking the electron micrographs.

To whom correspondence should be addressed.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Spencer, M.E., Hodge, R. Cloning and sequencing of the cDNA encoding the major albumin of Theobroma cacao . Planta 183, 528–535 (1991). https://doi.org/10.1007/BF00194274

Download citation

Key words

  • Albumin cDNA
  • Kunitz protease inhibitor
  • Theobroma