Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1984 Jun 11;12(11):4637–4652. doi: 10.1093/nar/12.11.4637

Expression of antigenic regions of a trypanosome variable surface glycoprotein in E. coli using Bal-31 nuclease digestion.

M J Lenardo, S T Brentano, J E Donelson
PMCID: PMC318864  PMID: 6204274

Abstract

We have used the expression of a trypanosome variable surface glycoprotein (VSG) in E. coli to produce VSG serotype-specific antisera which have none of the cross-reacting specificities characteristic of antisera prepared against purified VSGs. This was accomplished by treating restriction fragments of VSG cDNAs with Bal-31 nuclease to facilitate expression of their open reading frames in the E. coli expression vector, pMR100 (4). The resultant VSG-beta-galactosidase fusion proteins possess various antigenic regions of the original VSG. This provides a rapid means for producing VSG-specific antisera for reagent use and has the capability of large scale production of antigen for immunological investigation.

Full text

PDF
4637

Images in this article

4645Image
on p.4645
4646Image
on p.4646
4647Image
on p.4647

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Backman K., Ptashne M., Gilbert W. Construction of plasmids carrying the cI gene of bacteriophage lambda. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4174–4178. doi: 10.1073/pnas.73.11.4174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Backman K., Ptashne M. Maximizing gene expression on a plasmid using recombination in vitro. Cell. 1978 Jan;13(1):65–71. doi: 10.1016/0092-8674(78)90138-1. [DOI] [PubMed] [Google Scholar]
  3. Bolivar F., Backman K. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 1979;68:245–267. doi: 10.1016/0076-6879(79)68018-7. [DOI] [PubMed] [Google Scholar]
  4. Boothroyd J. C., Cross G. A., Hoeijmakers J. H., Borst P. A variant surface glycoprotein of Trypanosoma brucei synthesized with a C-terminal hydrophobic 'tail' absent from purified glycoprotein. Nature. 1980 Dec 11;288(5791):624–626. doi: 10.1038/288624a0. [DOI] [PubMed] [Google Scholar]
  5. Boothroyd J. C., Paynter C. A., Coleman S. L., Cross G. A. Complete nucleotide sequence of complementary DNA coding for a variant surface glycoprotein from Trypanosoma brucei. J Mol Biol. 1982 May 25;157(3):547–556. doi: 10.1016/0022-2836(82)90475-2. [DOI] [PubMed] [Google Scholar]
  6. Borst P., Cross G. A. Molecular basis for trypanosome antigenic variation. Cell. 1982 Jun;29(2):291–303. doi: 10.1016/0092-8674(82)90146-5. [DOI] [PubMed] [Google Scholar]
  7. Burgess R. R. Separation and characterization of the subunits of ribonucleic acid polymerase. J Biol Chem. 1969 Nov 25;244(22):6168–6176. [PubMed] [Google Scholar]
  8. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  9. Cross G. A. Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei. Parasitology. 1975 Dec;71(3):393–417. doi: 10.1017/s003118200004717x. [DOI] [PubMed] [Google Scholar]
  10. Crowe J. S., Barry J. D., Luckins A. G., Ross C. A., Vickerman K. All metacyclic variable antigen types of Trypanosoma congolense identified using monoclonal antibodies. Nature. 1983 Nov 24;306(5941):389–391. doi: 10.1038/306389a0. [DOI] [PubMed] [Google Scholar]
  11. Donelson J. E., Murphy W. J., Brentano S. T., Rice-Ficht A. C., Cain G. D. Comparison of the expression-linked extra copy (ELC) and basic copy (BC) genes of a trypanosome surface antigen. J Cell Biochem. 1983;23(1-4):1–12. doi: 10.1002/jcb.240230102. [DOI] [PubMed] [Google Scholar]
  12. Esser K. M., Schoenbechler M. J., Gingrich J. B. Trypanosoma rhodesiense blood forms express all antigen specificities relevant to protection against metacyclic (insect form) challenge. J Immunol. 1982 Oct;129(4):1715–1718. [PubMed] [Google Scholar]
  13. Gray M. R., Colot H. V., Guarente L., Rosbash M. Open reading frame cloning: identification, cloning, and expression of open reading frame DNA. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6598–6602. doi: 10.1073/pnas.79.21.6598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guarente L., Lauer G., Roberts T. M., Ptashne M. Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit beta-globin. Cell. 1980 Jun;20(2):543–553. doi: 10.1016/0092-8674(80)90640-6. [DOI] [PubMed] [Google Scholar]
  15. Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. doi: 10.1016/0076-6879(83)01013-7. [DOI] [PubMed] [Google Scholar]
  16. Guo L. H., Yang R. C., Wu R. An improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid. Nucleic Acids Res. 1983 Aug 25;11(16):5521–5540. doi: 10.1093/nar/11.16.5521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hajduk S., Vickerman K. Antigenic differentiation of Trypanosoma brucei: studies on metacyclic and first parasitaemia populations. Trans R Soc Trop Med Hyg. 1981;75(1):145–146. doi: 10.1016/0035-9203(81)90047-x. [DOI] [PubMed] [Google Scholar]
  18. Holder A. A., Cross G. A. Glycopeptides from variant surface glycoproteins of Trypanosoma Brucei. C-terminal location of antigenically cross-reacting carbohydrate moieties. Mol Biochem Parasitol. 1981 Feb;2(3-4):135–150. doi: 10.1016/0166-6851(81)90095-5. [DOI] [PubMed] [Google Scholar]
  19. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Lagrimini L. M., Brentano S. T., Donelson J. E. A DNA sequence analysis package for the IBM personal computer. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):605–614. doi: 10.1093/nar/12.1part2.605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lerner R. A., Green N., Alexander H., Liu F. T., Sutcliffe J. G., Shinnick T. M. Chemically synthesized peptides predicted from the nucleotide sequence of the hepatitis B virus genome elicit antibodies reactive with the native envelope protein of Dane particles. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3403–3407. doi: 10.1073/pnas.78.6.3403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  24. Morrison D. A. Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol. 1979;68:326–331. doi: 10.1016/0076-6879(79)68023-0. [DOI] [PubMed] [Google Scholar]
  25. Parsons M., Smit J., Nelson R. G., Stuart K., Agabian N. Expression of a Trypanosoma brucei brucei variant antigen in Escherichia coli. Mol Biochem Parasitol. 1984 Feb;10(2):207–216. doi: 10.1016/0166-6851(84)90008-2. [DOI] [PubMed] [Google Scholar]
  26. Pestka S. The human interferons--from protein purification and sequence to cloning and expression in bacteria: before, between, and beyond. Arch Biochem Biophys. 1983 Feb 15;221(1):1–37. doi: 10.1016/0003-9861(83)90118-2. [DOI] [PubMed] [Google Scholar]
  27. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  28. Rose M., Botstein D. Construction and use of gene fusions to lacZ (beta-galactosidase) that are expressed in yeast. Methods Enzymol. 1983;101:167–180. doi: 10.1016/0076-6879(83)01012-5. [DOI] [PubMed] [Google Scholar]
  29. Rüther U., Koenen M., Sippel A. E., Müller-Hill B. Exon cloning: immunoenzymatic identification of exons of the chicken lysozyme gene. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6852–6855. doi: 10.1073/pnas.79.22.6852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schoemaker J. M., Markovitz A. Identification of the gene lon (capR) product as a 94-kilodalton polypeptide by cloning and deletion analysis. J Bacteriol. 1981 Jul;147(1):46–56. doi: 10.1128/jb.147.1.46-56.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Smith G. E., Summers M. D. The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl-paper. Anal Biochem. 1980 Nov 15;109(1):123–129. doi: 10.1016/0003-2697(80)90019-6. [DOI] [PubMed] [Google Scholar]
  32. Smith H. O. Recovery of DNA from gels. Methods Enzymol. 1980;65(1):371–380. doi: 10.1016/s0076-6879(80)65048-4. [DOI] [PubMed] [Google Scholar]
  33. Talmadge K., Gilberg W. Construction of plasmid vectors with unique PstI cloning sites in a signal sequence coding region. Gene. 1980 Dec;12(3-4):235–241. doi: 10.1016/0378-1119(80)90105-5. [DOI] [PubMed] [Google Scholar]
  34. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Weinstock G. M., ap Rhys C., Berman M. L., Hampar B., Jackson D., Silhavy T. J., Weisemann J., Zweig M. Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4432–4436. doi: 10.1073/pnas.80.14.4432. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES