A fusion protein library: an improved method for rapid screening and characterization of DNA binding or interacting proteins.
A rapid method for screening and characterization of DNA binding or protein-interacting molecules is described. The method relies on a fusion protein library in which randomized DNA fragments are inserted into pGEX-3X and its derivatives to generate collections of GST-fusion proteins. After inducing the expression of the fusion proteins by addition of IPTG, the colonies can be screened either with radioactively labeled DNA/RNA fragment for specific clones encoding DNA/RNA binding proteins or with an antibody for clones encoding proteins of interest. They can also be screened with a radioactively labeled protein for cloning of interacting molecules. The fusion proteins encoded by the isolated clones can be readily purified by conducting the lysis of the cells and an affinity column in the presence of an alkyl anionic detergent, N-laurylsarcosine (sarkosyl), and can be further characterized.
Ikeda
M
M
Department of Molecular and Developmental Biology, University of Tokyo, Japan.
Arai
K
K
Masai
H
H
D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics; D015723:Gene Library; D005982:Glutathione Transferase / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012680:Sensitivity and Specificity
D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase