Prophage lambda libraries for isolating cDNA clones by functional screening.
Isolation of cDNA clones from lambda gt11 phage libraries by functional screening is limited by the low amount of lacZ-cDNA-encoded fusion protein synthesized in an isolated phage plaque. The amount of specific cDNA-encoded protein can be significantly enhanced by expression in bacterial colonies rather than phage plaques. Escherichia coli was lysogenized with a lambda gt11 cDNA expression library from Dictyostelium discoideum. Bacteria were selected for the presence of the lambda gt11 prophage by elimination of nonlysogenic parental cells with a lambda cI phage. The usefulness of the lysogen library was demonstrated by immuno-screening and functional screening with two different radiolabeled ligands. cDNA clones encoding a well-characterized D. discoideum protein, the regulatory subunit of the cAMP-dependent protein kinase, were isolated by screening the lysogen library with antibodies. Clones encoding this protein could also be identified by functional screening with [3H]cAMP, demonstrating that the limit of detection of positive clones by ligand screening is at least an order of magnitude lower for the lysogen library than for the corresponding phage library. We have subsequently used the lysogen library to isolate cDNA clones encoding calmodulin-binding protein(s) from D. discoideum by functional screening with [125I]calmodulin. For these clones, screening of the corresponding phage library had previously been found unsuccessful.
Mutzel
R
R
Fakultät für Biologie, Universität Konstanz, F.R.G.
Baeuerle
A
A
Jung
S
S
Dammann
H
H
D010582:Bacteriophage lambda / Q000235:genetics*; D002148:Calmodulin-Binding Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification; D004023:Dictyostelium / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005814:Genes, Viral; D008242:Lysogeny; D011494:Protein Kinases / Q000235:genetics
D002148:Calmodulin-Binding Proteins; D004279:DNA, Viral; D004247:DNA; D011494:Protein Kinases