Part of the Methods in Molecular Biology™ book series (MIMB, volume 81)
Expression Library Screening
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).
KeywordsGlycerol Agar Electrophoresis Polyacrylamide Dodecyl
- 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.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.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
© Humana Press Inc. 1998