Screening libraries to identify proteins with desired binding activities using a split-GFP reassembly assay.
Designer protein modules, which bind specifically to a desired target, have numerous potential applications. One approach to creating such proteins is to construct and screen libraries. Here we present a detailed description of using a split-GFP reassembly assay to screen libraries and identify proteins with novel binding properties. Attractive features of the split-GFP based screen are the absence of false positives and the simplicity, robustness, and ease of automation of the screen. Here, we describe both the construction of a naive protein library, and screening of the library using the split-GFP assay to identify proteins that bind specifically to chosen peptide sequences.
Jackrel
Meredith E
ME
Departments of Chemistry, Yale University, New Haven, Connecticut 06520, USA.
Cortajarena
Aitziber L
AL
Liu
Tina Y
TY
Regan
Lynne
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D019151:Peptide Library; D011506:Proteins; D049452:Green Fluorescent Proteins