Screening Peptide/Protein Libraries Fused to the λ Repressor DNA-Binding Domain in E. coli Cells

The use of λ repressor fusions to study protein-protein interactions in E. coli was first described by Hu and others (1). Since then, the repressor system has been employed by several laboratories to screen genomic (2–5) and cDNA libraries (6) for homotypic or heterotypic interactions. λ repressor consists of distinct and separable domains: the N-terminal domain which has DNA binding activity and the C-terminal domain which mediates dimerization. The repressor fusion system is based on reconstituting the activity of the repressor by replacing the C-terminal domain with a heterologous oligomerization domain. The interaction is detected when the C-terminal domain forms a dimer (or higher order oligomer) with itself (homotypic interaction) or with a different domain from other fusion (heterotypic interaction) (see Fig. 1). Fig. 1 The rationale of λ repressor fusions. Repressor fusions are used to detect protein-protein interactions in vivo. Protein or peptide targets are fused to the λ repressor DNA binding domain; these fusions can be evaluated for repressor activity ... Repressor fusions are usually expressed from multicopy plasmids; for a detailed discussion of repressor fusion plasmids available from our laboratory see ref. 7. Similar plasmids have been constructed by other groups (5,8–10) with a variety of modifications. In all cases, unique restriction sites are available for cloning a desired insert in-frame with the N-terminal domain of repressor. Table 1 lists the features of several of the repressor plasmid vectors in the literature. Table 1 Repressor Fusion Vectors Used for Peptide/Protein Lobrary Screening The identification and characterization of homotypic or heterotypic interactions is done by fusing a target DNA (fragments from a specific gene of interest, or a genomic, cDNA, randomized, or rationally designed library) to the λ repressor DNA binding domain. Repressor fusion libraries are made by using appropriate vectors with standard cloning methods. Library construction is not discussed further in this chapter (see Note 1). Here, we focus on the evaluation of the resultant repressor fusions for repressor activity using either immunity to phage infection (see Subheading 3.1.) or a variety of reporters under λ repressor control (see Table 2 and Subheadings 3.2–3.4.). Further screening is useful to ensure that the repressor activity of the fusion protein is dependent on the insert, especially when evaluating clones isolated by selection. A simple a high-throughput screening strategy based on nonsense suppression is described in Subheading 3.5. Table 2 Reporters Available for Library Screening Using Repressor Fusions 2. Materials Different subsets of the materials listed below are needed for the different protocols 2.1. General Use Media, Antibiotics, and Materials Luria-Bertani (LB) broth and agar: Premixed LB broth (DIFCO, cat. no. 244620) and agar (DIFCO, cat. no. 244620) are prepared according to the vendors instructions. 2XYT broth per L: 16 g tryptone, 10 g yeast extract, 10 g NaCl. Dissolve in 1 L distilled H2O. Autoclave. Antibiotics: Ampicillin 200 mg/mL in H2O (1000X stock, use at a final concentration of 200 μg/mL); kanamycin 20 mg/mL in H2O (1000X stock, use at a final concentration of 20 μg/mL). Sterile 96-well microplates (clinical “V bottom”). Microplate replicator 96 pin (Boekel Model 140500). Multichannel pipetter (8 or 12-channel) to handle volumes from 5–200 μL. Sterile toothpicks. 2.2. Strains Strains used are listed in Table 3. Different strains are used for each of the screening approaches described below. Table 3 E. coli Strains Used for Peptide/Protein Library Selection and Screening 2.3. For Phage Immunity Selections and Screens AG1688 and JH787 (see Note 2). λKH54 and λKH54h80 phage stocks at 109–1010 plaque forming units (pfu)/mL (see Note 3). Tryptone broth per L: 10 g Tryptone, 5 g NaCl. Dissolve in 1 L H2O. Autoclave. Tryptone agar: 13 g Bacto-Agar/L of tryptone broth before autoclaving. Tris-Magnesium (TM) buffer: 10 mM Tris-HCl, pH 8.0, 10 mM MgSO4. Autoclave. Tryptone top agar: 0.7 g Bacto agar/100 mL of tryptone broth before autoclaving. Chloroform. 15-cm LB plates containing ampicillin and kanamycin (see Note 4). 100-mm LB Amp Kan plates containing 25 mM sodium citrate, added from a sterile 1M stock solution. 2.4. For Screening with lacZ-Based Reporters Materials for β-galactosidase assay of choice (11).