Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1991 Nov 15;280(Pt 1):219–224. doi: 10.1042/bj2800219

Biologically active and amidated cecropin produced in a baculovirus expression system from a fusion construct containing the antibody-binding part of protein A.

D Andersons 1, A Engström 1, S Josephson 1, L Hansson 1, H Steiner 1
PMCID: PMC1130623  PMID: 1720614

Abstract

A synthetic antibody-binding part derived from protein A from Staphylococcus aureus was used as a fusion partner in a eukaryotic expression system employing Autographa californica nuclear polyhedrosis as a vector. This, in conjunction with an efficient signal sequence, facilitated the purification of the antibacterial peptide cecropin A from the medium of Spodoptera frugiperda cells infected with a recombinant virus. In order to increase further the concentrations of fusion protein, Trichoplusia ni larvae were used as host. Cecropin A could be obtained after cleavage of the fusion protein with CNBr. Biological activity as well as the correct structure including the C-terminal amide group was shown using electrophoresis with detection of antibacterial proteins and mass spectroscopy.

Full text

PDF
219

Images in this article

220Fig. 1.
on p.220
221Fig. 2.
on p.221
222Fig. 3.
on p.222
222Fig. 4.
on p.222

Selected References

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

  1. Andreu D., Merrifield R. B., Steiner H., Boman H. G. Solid-phase synthesis of cecropin A and related peptides. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6475–6479. doi: 10.1073/pnas.80.21.6475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boman H. C., Boman I. A., Andreu D., Li Z. Q., Merrifield R. B., Schlenstedt G., Zimmermann R. Chemical synthesis and enzymic processing of precursor forms of cecropins A and B. J Biol Chem. 1989 Apr 5;264(10):5852–5860. [PubMed] [Google Scholar]
  3. Boman H. G., Hultmark D. Cell-free immunity in insects. Annu Rev Microbiol. 1987;41:103–126. doi: 10.1146/annurev.mi.41.100187.000535. [DOI] [PubMed] [Google Scholar]
  4. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  5. Goeddel D. V., Kleid D. G., Bolivar F., Heyneker H. L., Yansura D. G., Crea R., Hirose T., Kraszewski A., Itakura K., Riggs A. D. Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci U S A. 1979 Jan;76(1):106–110. doi: 10.1073/pnas.76.1.106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hellers M., Gunne H., Steiner H. Expression of post-translational processing of preprocecropin A using a baculovirus vector. Eur J Biochem. 1991 Jul 15;199(2):435–439. doi: 10.1111/j.1432-1033.1991.tb16141.x. [DOI] [PubMed] [Google Scholar]
  7. Hink W. F. Established insect cell line from the cabbage looper, Trichoplusia ni. Nature. 1970 May 2;226(5244):466–467. doi: 10.1038/226466b0. [DOI] [PubMed] [Google Scholar]
  8. Hultmark D., Engström A., Bennich H., Kapur R., Boman H. G. Insect immunity: isolation and structure of cecropin D and four minor antibacterial components from Cecropia pupae. Eur J Biochem. 1982 Sep;127(1):207–217. doi: 10.1111/j.1432-1033.1982.tb06857.x. [DOI] [PubMed] [Google Scholar]
  9. Hultmark D., Steiner H., Rasmuson T., Boman H. G. Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur J Biochem. 1980 May;106(1):7–16. doi: 10.1111/j.1432-1033.1980.tb05991.x. [DOI] [PubMed] [Google Scholar]
  10. Itakura K., Hirose T., Crea R., Riggs A. D., Heyneker H. L., Bolivar F., Boyer H. W. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science. 1977 Dec 9;198(4321):1056–1063. doi: 10.1126/science.412251. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Lebacq-Verheyden A. M., Kasprzyk P. G., Raum M. G., Van Wyke Coelingh K., Lebacq J. A., Battey J. F. Posttranslational processing of endogenous and of baculovirus-expressed human gastrin-releasing peptide precursor. Mol Cell Biol. 1988 Aug;8(8):3129–3135. doi: 10.1128/mcb.8.8.3129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Luckow V. A., Summers M. D. High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors. Virology. 1989 May;170(1):31–39. doi: 10.1016/0042-6822(89)90348-6. [DOI] [PubMed] [Google Scholar]
  14. Löwenadler B., Jansson B., Paleus S., Holmgren E., Nilsson B., Moks T., Palm G., Josephson S., Philipson L., Uhlén M. A gene fusion system for generating antibodies against short peptides. Gene. 1987;58(1):87–97. doi: 10.1016/0378-1119(87)90032-1. [DOI] [PubMed] [Google Scholar]
  15. Maeda S. Increased insecticidal effect by a recombinant baculovirus carrying a synthetic diuretic hormone gene. Biochem Biophys Res Commun. 1989 Dec 29;165(3):1177–1183. doi: 10.1016/0006-291x(89)92726-5. [DOI] [PubMed] [Google Scholar]
  16. Moks T., Abrahmsén L., Holmgren E., Bilich M., Olsson A., Uhlén M., Pohl G., Sterky C., Hultberg H., Josephson S. Expression of human insulin-like growth factor I in bacteria: use of optimized gene fusion vectors to facilitate protein purification. Biochemistry. 1987 Aug 25;26(17):5239–5244. doi: 10.1021/bi00391a005. [DOI] [PubMed] [Google Scholar]
  17. Nilsson B., Moks T., Jansson B., Abrahmsén L., Elmblad A., Holmgren E., Henrichson C., Jones T. A., Uhlén M. A synthetic IgG-binding domain based on staphylococcal protein A. Protein Eng. 1987 Feb-Mar;1(2):107–113. doi: 10.1093/protein/1.2.107. [DOI] [PubMed] [Google Scholar]
  18. Steiner H., Andreu D., Merrifield R. B. Binding and action of cecropin and cecropin analogues: antibacterial peptides from insects. Biochim Biophys Acta. 1988 Apr 7;939(2):260–266. doi: 10.1016/0005-2736(88)90069-7. [DOI] [PubMed] [Google Scholar]
  19. Steiner H., Hultmark D., Engström A., Bennich H., Boman H. G. Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature. 1981 Jul 16;292(5820):246–248. doi: 10.1038/292246a0. [DOI] [PubMed] [Google Scholar]
  20. Steiner H., Pohl G., Gunne H., Hellers M., Elhammer A., Hansson L. Human tissue-type plasminogen activator synthesized by using a baculovirus vector in insect cells compared with human plasminogen activator produced in mouse cells. Gene. 1988 Dec 20;73(2):449–457. doi: 10.1016/0378-1119(88)90509-4. [DOI] [PubMed] [Google Scholar]
  21. Steiner H. Secondary structure of the cecropins: antibacterial peptides from the moth Hyalophora cecropia. FEBS Lett. 1982 Jan 25;137(2):283–287. doi: 10.1016/0014-5793(82)80368-2. [DOI] [PubMed] [Google Scholar]
  22. Sun S. C., Lindström I., Boman H. G., Faye I., Schmidt O. Hemolin: an insect-immune protein belonging to the immunoglobulin superfamily. Science. 1990 Dec 21;250(4988):1729–1732. doi: 10.1126/science.2270488. [DOI] [PubMed] [Google Scholar]
  23. Yamada K., Nakajima Y., Natori S. Production of recombinant sarcotoxin IA in Bombyx mori cells. Biochem J. 1990 Dec 15;272(3):633–636. doi: 10.1042/bj2720633. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES