CIS display: In vitro selection of peptides from libraries of protein-DNA complexes

Abstract

We describe the development of an in vitro library selection system (CIS display) that exploits the ability of a DNA replication initiator protein (RepA) to bind exclusively to the template DNA from which it has been expressed, a property called cis-activity. A diverse peptide library is created by ligation of DNA fragments of random sequence to a DNA fragment that encodes RepA. After in vitro transcription and translation, a pool of protein-DNA complexes is formed where each protein is stably associated with the DNA that encodes it. These complexes are amenable to the affinity selection of ligands to targets of interest. Here we show that RepA is a highly faithful cis-acting DNA-binding protein and demonstrate that libraries encoding >10(12) random 18-mer peptides can be constructed and used to isolate peptides that bind specifically to disparate targets. The use of DNA to encode the displayed peptides offers advantages over in vitro peptide display systems that use mRNA.

Fig. 1.

The principle of the CIS display technology. (A) Template DNA encoding an N-terminal library peptide is ligated to the RepA gene. In vitro transcription is initiated at the promoter and pauses when the RNA polymerase reaches the CIS element. Concurrent translation produces the RepA protein, which transiently interacts with the CIS element, thereby forcing its subsequent binding to the adjacent ori sequence. This process establishes a faithful linkage between a template DNA and the expressed polypeptide that it encodes. (B) CIS display selections begin with the construction of a peptide-encoding DNA library followed by in vitro transcription/translation to form a pool of protein–DNA complexes (step 1). The library pool is incubated with an immobilized target (step 2), nonbinding peptides are washed away (step 3), and the retained DNA that encodes the target-binding peptides is eluted and amplified by PCR (step 4), to form a DNA library ready for the next round of selection. After three to five rounds of selection, recovered DNA is cloned into an appropriate expression vector for the identification of individual target-binding peptide sequences.

Fig. 2.

Demonstration of target-specific ligand selection by using CIS display. (A) A 1:1 mixture of Cκ-RepA DNA and V5-repA DNA was prepared, transcribed and translated in vitro, and selected against either anti-Ck antibody, anti-V5 antibody, or anti-ACTH antibody. Recovered DNA was amplified with universal primers and separated by agarose gel electrophoresis on the basis of the size difference between the Cκ domain and the V5 peptide tag. The amount of DNA specifically recovered when by using the anti-Cκ and anti-V5 antibodies is shown in duplicate along with a single negative control (anti-ACTH) to reflect background recovery. (B) A 1:10⁸ dilution of Cκ-RepA DNA into V5-repA DNA was prepared and subjected to four rounds of selection against either the anti-Cκ antibody or the anti-FLAG antibody as a negative control. The four rounds of selection are indicated along with the positions of the two recovered PCR products. (C) A 1:10¹⁰ dilution of Cκ-RepA DNA into V5-repA DNA was prepared and subjected to four rounds of selection against either the anti-Cκ antibody or the anti-FLAG antibody as a negative control. The four rounds of selection are indicated along with the positions of the two recovered PCR products. Marker refers to the DNA Hyperladder (Bioline, London).

Fig. 3.

Homology between selected binding peptides and respective epitope sequences. The sequences of 11 DO1 (A) and 23 M2-binding peptides (B) are aligned with the P53-DO1 and FLAG-M2 epitope sequences, respectively. Clones from the DO1 selection are taken from round 5, and the M2 peptides originate from rounds 4 and 5. Regions of homology are shaded, and the frequency with which each clone was identified is shown in parentheses.

Fig. 4.

ELISA of peptides selected against lysozyme both on solid-phase and in-solution selection. After five rounds of selection, peptides expressed on phage as M13 gpVIII fusions were checked for specificity of binding to lysozyme (50 μg/ml), ovalbumin (50 μg/ml), Bcl-X (10 μg/ml), BSA (200 μg/ml), and anti-V5 antibody (10 μg/ml). Two examples of the selected clones are shown; E6 and F11, from solid-phase and in-solution selection, respectively. The V5 peptide fused to gpVIII was used as a both a negative control (against lysozyme) and a positive control (against anti-V5 antibody) for the assay.