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. 1998 Apr 1;26(7):1718–1723. doi: 10.1093/nar/26.7.1718

Baculovirus surface display: construction and screening of a eukaryotic epitope library.

W Ernst 1, R Grabherr 1, D Wegner 1, N Borth 1, A Grassauer 1, H Katinger 1
PMCID: PMC147480  PMID: 9512544

Abstract

The baculovirus expression system was utilized to serve as a tool for ligand selection, demonstrating the applicability of the system to the generation and screening of eukaryotic expression libraries. The HIV-1-gp41 epitope 'ELDKWA', specific for the neutralizing human mAb 2F5, was inserted into the antigenic site B of influenza virus hemagglutinin and expressed on the surface of baculovirus infected insect cells. In order to improve the antigenicity of the epitope within the hemagglutinin, and therefore enhance the specific binding of 2F5, we inserted three additional, random amino acids adjacent to the epitope. This pool of hemagglutinin genes was directly cloned into the baculovirus Ac-omega. To identify distinct proteins displayed on the cellular surface, we developed a screening protocol to select for specific binding capacity of individual viral clones. Using fluorescence activated cell sorting (FACS) we isolated a baculovirus clone displaying the epitope with markedly increased binding capacity out of a pool of 8000 variants in only one sorting step. Binding properties of the identified ligand were examined by FACS performing a competition assay.

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Selected References

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  1. Boder E. T., Wittrup K. D. Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol. 1997 Jun;15(6):553–557. doi: 10.1038/nbt0697-553. [DOI] [PubMed] [Google Scholar]
  2. Boublik Y., Di Bonito P., Jones I. M. Eukaryotic virus display: engineering the major surface glycoprotein of the Autographa californica nuclear polyhedrosis virus (AcNPV) for the presentation of foreign proteins on the virus surface. Biotechnology (N Y) 1995 Oct;13(10):1079–1084. doi: 10.1038/nbt1095-1079. [DOI] [PubMed] [Google Scholar]
  3. Buchacher A., Predl R., Strutzenberger K., Steinfellner W., Trkola A., Purtscher M., Gruber G., Tauer C., Steindl F., Jungbauer A. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Res Hum Retroviruses. 1994 Apr;10(4):359–369. doi: 10.1089/aid.1994.10.359. [DOI] [PubMed] [Google Scholar]
  4. Domingo D. L., Trowbridge I. S. Characterization of the human transferrin receptor produced in a baculovirus expression system. J Biol Chem. 1988 Sep 15;263(26):13386–13392. [PubMed] [Google Scholar]
  5. Ernst W. J., Grabherr R. M., Katinger H. W. Direct cloning into the Autographa californica nuclear polyhedrosis virus for generation of recombinant baculoviruses. Nucleic Acids Res. 1994 Jul 25;22(14):2855–2856. doi: 10.1093/nar/22.14.2855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Georgiou G., Stathopoulos C., Daugherty P. S., Nayak A. R., Iverson B. L., Curtiss R., 3rd Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines. Nat Biotechnol. 1997 Jan;15(1):29–34. doi: 10.1038/nbt0197-29. [DOI] [PubMed] [Google Scholar]
  7. Ghiasi H., Nesburn A. B., Wechsler S. L. Cell surface expression of herpes simplex virus type 1 glycoprotein H in recombinant baculovirus-infected cells. Virology. 1991 Nov;185(1):187–194. doi: 10.1016/0042-6822(91)90766-5. [DOI] [PubMed] [Google Scholar]
  8. Grabherr R., Ernst W., Doblhoff-Dier O., Sara M., Katinger H. Expression of foreign proteins on the surface of Autographa californica nuclear polyhedrosis virus. Biotechniques. 1997 Apr;22(4):730–735. doi: 10.2144/97224rr02. [DOI] [PubMed] [Google Scholar]
  9. Groebe D. R., Chung A. E., Ho C. Cationic lipid-mediated co-transfection of insect cells. Nucleic Acids Res. 1990 Jul 11;18(13):4033–4033. doi: 10.1093/nar/18.13.4033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hoogenboom H. R., Winter G. By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J Mol Biol. 1992 Sep 20;227(2):381–388. doi: 10.1016/0022-2836(92)90894-p. [DOI] [PubMed] [Google Scholar]
  11. Kerschbaumer R. J., Hirschl S., Schwager C., Ibl M., Himmler G. pDAP2: a vector for construction of alkaline phosphatase fusion-proteins. Immunotechnology. 1996 Jun;2(2):145–150. doi: 10.1016/1380-2933(96)00040-1. [DOI] [PubMed] [Google Scholar]
  12. Kuroda K., Hauser C., Rott R., Klenk H. D., Doerfler W. Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector. EMBO J. 1986 Jun;5(6):1359–1365. doi: 10.1002/j.1460-2075.1986.tb04367.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kuroda K., Veit M., Klenk H. D. Retarded processing of influenza virus hemagglutinin in insect cells. Virology. 1991 Jan;180(1):159–165. doi: 10.1016/0042-6822(91)90019-8. [DOI] [PubMed] [Google Scholar]
  14. Li S., Polonis V., Isobe H., Zaghouani H., Guinea R., Moran T., Bona C., Palese P. Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1. J Virol. 1993 Nov;67(11):6659–6666. doi: 10.1128/jvi.67.11.6659-6666.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McLinden J. H., Ploplis V. A., Fraser M. J., Rosen E. D. Expression of foreign epitopes on recombinant occlusion bodies of baculoviruses. Vaccine. 1992;10(4):231–237. doi: 10.1016/s0264-410x(06)80001-0. [DOI] [PubMed] [Google Scholar]
  16. Mottershead D., van der Linden I., von Bonsdorff C. H., Keinänen K., Oker-Blom C. Baculoviral display of the green fluorescent protein and rubella virus envelope proteins. Biochem Biophys Res Commun. 1997 Sep 29;238(3):717–722. doi: 10.1006/bbrc.1997.7372. [DOI] [PubMed] [Google Scholar]
  17. Muster T., Guinea R., Trkola A., Purtscher M., Klima A., Steindl F., Palese P., Katinger H. Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS. J Virol. 1994 Jun;68(6):4031–4034. doi: 10.1128/jvi.68.6.4031-4034.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Possee R. D. Cell-surface expression of influenza virus haemagglutinin in insect cells using a baculovirus vector. Virus Res. 1986 Jul;5(1):43–59. doi: 10.1016/0168-1702(86)90064-x. [DOI] [PubMed] [Google Scholar]
  19. Scott J. K., Smith G. P. Searching for peptide ligands with an epitope library. Science. 1990 Jul 27;249(4967):386–390. doi: 10.1126/science.1696028. [DOI] [PubMed] [Google Scholar]
  20. Vaughan T. J., Williams A. J., Pritchard K., Osbourn J. K., Pope A. R., Earnshaw J. C., McCafferty J., Hodits R. A., Wilton J., Johnson K. S. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol. 1996 Mar;14(3):309–314. doi: 10.1038/nbt0396-309. [DOI] [PubMed] [Google Scholar]
  21. Winter G., Griffiths A. D., Hawkins R. E., Hoogenboom H. R. Making antibodies by phage display technology. Annu Rev Immunol. 1994;12:433–455. doi: 10.1146/annurev.iy.12.040194.002245. [DOI] [PubMed] [Google Scholar]

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