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Expression Library Screening

  • Gary D. Foster
Part of the Methods in Molecular Biology™ book series (MIMB, volume 81)

Abstract

When trying to identify a clone within a cDNA library, which may contain a coat protein (CP) gene, one useful technique may by immunological screening, using antibodies raised against either purified virus or isolated CP. Antibody screening can be carried out on a cDNA cloned into a wide range of vectors, including plasmids and phage-based vectors. Indeed, a whole plethora of commercial vectors are now available that have been optimized for generating expression libraries, including λ-gt11, λZAP (Stratagene, La Jolla, CA). However, antibody screening can be carried out on the simplest of plasmid vectors, based on the principle that, if the plasmid uses blue/white color selection, then a percentage of the cDNA inserts will be expressed as a fusion protein with β-galactosidase when the cells are induced with IPTG. The method described within this chapter will deal with such a plasmid screen, with readers directed to λ-screening chapters by Somssich and WeiBhaar in Plant Gene Isolation (1) and Hurst in cDNA Library Protocols (2), and (one of the original and best descriptions) by Huynh et al. 3 in DNA Cloning: A Practical Approach (3), all being good references for suitable lambda protocols. A typical immunological screen is shown in Fig. 1, for a pUC13 vector (4). Double-stranded cDNA to the carlavirus, Helenium virus S (HelVS) was ligated into SmaI digested pUC13 vector and transformed into competent Escherichia coli. Colonies were screened with both nucleic acid probes using HelVS specific (32P) first-strand cDNA (Fig. 1 A) and also using HelVS polyclonal antisera (Fig. 1 B).
Fig. 1.

(A) Colony hybridization using HelVS specific (32P) cDNA. Position 28 represents pUC 13 nonrecombinant control. (B) Colony hybridization using HelVS polyclonal antisera. Colonies were streaked onto nitrocellulose (top panel) and grown overnight prior to screening. Note that color development is evident on the reverse side on which colonies were streaked (bottom panel). Colony 28 represents pUCl3 nonrecombinant control. (C) Western blot analysis of clones expressing HelVS coat protein. Lanes 19′, 23′, and CP′: Coomassie blue stained gel of bacterial lysates and HelVS CP. Lanes 19, 23, and CP: Western blot analysis of bacterial lysates and HelVS CP reacted with HelVS polyclonal antisera. Positions of the CP-related products are indicated with arrows. No signals were obtained in Western blots from untransformed bacterial cell lysates.

Keywords

Coat Protein Gentle Agitation pUC13 Vector Antibody Screening Nucleic Acid Probe 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Somssich, I. E. and WeiBhaar, B. (1996) Expressing library screening, in Plant Gene Isolation (Foster, G. D. and Twell, D., eds.), Wiley, New York.Google Scholar
  2. 2.
    Hurst, H. C. (1997) Immunological screening of phage cDNA expression libraries, in cDNA Library Protocols (Cowell, I. G. and Austin, A. A., eds.), Humana, Totowa, NJ, pp. 155–159.Google Scholar
  3. 3.
    Huynh, T. V., Young, R. A., and Davies, R. W. (1988) Constructing and screening cDNA libraries in lambda-gt10 and lambda-gt11, in DNA Cloning: A practical Approach, vol 1. (Glover, D. M., ed.), IRL, Oxford, UK, pp. 49–78.Google Scholar
  4. 4.
    Foster, G. D., Scott, R., Draper, J., and Mills, P. R. (1992) Expression of Helenium virus S coat protein in Escherichia coli, in vitro in rabbit reticulocyte and transgenic tobacco. Acta Virol. 36, 567–575.PubMedGoogle Scholar
  5. 5.
    Foster, G. D., Millar, A. W., Meehan, B. M., and Mills, P. R. (1990) Nucleotide sequence of the 3-terminal region of Helenium virus S RNA. J. Gen. Virol. 71, 1877–1880.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1998

Authors and Affiliations

  • Gary D. Foster
    • 1
  1. 1.Department of BotanyUniversity of LeicesterUK

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