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 Sma I digested pUC13 vector and transformed into competent Escherichia coli . Colonies were screened with both nucleic acid probes using HelVS specific (³² P) first-strand cDNA (Fig. 1 A ) and also using HelVS polyclonal antisera (Fig. 1 B ).