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.

The P2-like coliphages are highly similar; the structural genes show at least 96% identity. However, at two loci they have genes believed to be horizontally transferred. We show that the genetic content at the second loci, the TO region, contains six completely different sequences with high AT contents and with different open reading frames. The product of one of them exhibits reverse transcriptase activity and blocks infection of phage T5.Temperate coliphage P2 has three nonessential lysogenic conversion genes, Z/fun, tin, and old, conferring resistance to infections by phage T5, T-even phages, and lambda phages, respectively (1,8,11,12,15,21). These genes have been shown to have a higher AT content than the rest of the genome and a codon usage that differs from that of the host, which suggests that they are horizontally transferred genes (3). P2-like prophages are common in Escherichia coli; about 30% of the ECOR collection (20) contain P2-like prophages. At the region equivalent to the P2 Z/fun gene, i.e., the Z region, which is located between the well-conserved tail genes G and F I , these P2-like prophages have been shown to contain different gene cassettes surrounded by a highly similar inverted repeat, indicating a site-specific integration event. Similar inverted repeats, spacing other genes, can be found in genetically unstable regions in pathogenic enterobacteria (19). The tin and old genes are located at the right end of the P2 genetic map close to the cos site. The old gene encodes an exonuclease that blocks multiplication of lambdoid phages (16), and tin encodes a protein that inhibits T4 DNA synthesis by poisoning the T4 single-stranded binding protein (15). These lysogenic conversion genes have an unusual AT content and codon utilization and are believed to have been additions from foreign genomes. This is supported by the fact that genes for hypothetical proteins similar to Old are found in various bacterial genomes in the UniProt GenBank (Table 1), while the Tin protein so far is unique. Furthermore, P2-related phages found in other enterobacteria contain other genes at this locus (4-6, 14, 17, 18). To clarify the nature of this locus in the P2-like coliphages, we have sequenced and characterized the region equivalent to the P2 tin and old genes, i.e., the TO region, in seven of the P2-like prophages in the ECOR library.Sequence variation between the A genes and the cos sites of the prophages. To sequence the region located between the A genes and the cos sites of the prophages, DNA was extracted from seven strains of the ECOR collection, which are known to contain P2-like prophages (P2-ECnb). A primer located at the catalytic site of the A gene and a primer located to the left of the cos sequence were used for the PCR amplifications, and specific amplified DNA fragments of variable length were obtained. The fragments were either sequenced directly or first cloned and then sequenced. To obtain the complete region, plasmid primers and internal primers were used. All strains contained different D...

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Botulinum neurotoxins are highly toxic substances and are all encoded together with one of two alternative gene clusters, the HA or the OrfX gene cluster. Very little is known about the function and structure of the proteins encoded in the OrfX gene cluster, which in addition to the toxin contains five proteins (OrfX1, OrfX2, OrfX3, P47, and NTNH). We here present the structures of OrfX2 and P47, solved to 2.1 and 1.8 Å, respectively. We show that they belong to the TULIP protein superfamily, which are often involved in lipid binding. OrfX1 and OrfX2 were both found to bind phosphatidylinositol lipids.

One drawback to the use of peptides as therapeutics has been their susceptibility to proteolysis. Here, we have used an in vitro display technology, CIS display, to enhance the proteolytic resistance of ligand-binding peptides by selection of protecting motifs from a large peptide library. The premise to this selection was that certain linear peptides within a library could form structures capable of preventing the access of proteases to defined cleavage sites without affecting ligand binding. A diverse 12-mer peptide library was inserted between a FLAG epitope motif and a thrombin cleavage site and this construct was fused to the bacterial initiator protein RepA for CIS display selection. After five rounds of selection, protection motifs were isolated that were capable of preventing proteolytic cleavage of the adjacent thrombin site. Some of the selected peptides were also resistant to more promiscuous proteases, such as chymotrypsin and trypsin, which were not used in the selection. The observed resistance to thrombin, trypsin and chymotrypsin translated into increased resistance to plasma proteases in vitro and to an increase in circulating half-lives in rats. This method can be applied to enhancing the in vivo stability of therapeutic peptides.