cDNA library screening using the SOS recruitment system.
The SOS recruitment system (SRS), a recently developed method for detecting protein-protein interactions, provides an attractive alternative to identify biologically important protein interactions. In SRS, the protein-protein interactions take place in the cytoplasm instead of the nucleus, as is the case in the conventional two-hybrid system. Although the SRS has overcome some of the disadvantages of the conventional two-hybrid system, it still has several problems and limitations. Here, we describe a new protocol for SRS library screening. A new combination of growth media to avoid the tedious step of replica plating greatly increases the number of independent colonies in a single library screening. Furthermore, we designed a pair of ras-specific primers and a one-step simple PCR to rule out the most abundant false positive, the mammalian ras cDNA, in SRS library screening.
Huang
W
W
University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
Wang
S L
SL
Lozano
G
G
de Crombrugghe
B
B
D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000494:pharmacology; D015723:Gene Library; D005947:Glucose / Q000494:pharmacology; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D055758:SOX9 Transcription Factor; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D020664:Son of Sevenless Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D018631:ras Proteins / Q000378:metabolism
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D011993:Recombinant Fusion Proteins; D055758:SOX9 Transcription Factor; D020664:Son of Sevenless Proteins; D014157:Transcription Factors; D018631:ras Proteins; D005947:Glucose; D005690:Galactose