A method for efficient gene isolation from phage lambda gt11 libraries: use of antisera to denatured, acetone-precipitated proteins.
Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot. Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful. It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained. These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage. This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts. These results show that the efficiency of screening expression libraries in E. coli can be improved if antibodies are raised against denatured proteins.
D000096:Acetone; D000914:Antibodies, Viral; D010582:Bacteriophage lambda / Q000235:genetics*; D011232:Chemical Precipitation; D003001:Cloning, Molecular; D005814:Genes, Viral; D011558:Herpesvirus 1, Suid / Q000235:genetics / Q000276:immunology; D011489:Protein Denaturation; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
D000914:Antibodies, Viral; D014764:Viral Proteins; D000096:Acetone