Identification from a phage display library of peptides that bind to toxic shock syndrome toxin-1 and that inhibit its binding to major histocompatibility complex (MHC) class II molecules.

Phage display technique is a powerful tool with which to identify novel binding sequences for antibody and receptor targets. Few studies, however, have used this technology to select affinity peptides for ligand molecules. Here, we screened a peptide phage library for binding to toxic shock syndrome toxin 1 (TSST-1) to examine whether peptide ligands for TSST-1 which mimic the structure of major histocompatibility complex (MHC) class II receptors could be identified. After three cycles of biopanning, four potent sequences reactive with TSST-1 were isolated (designated phages 2, 3, 8, and 11). Selected phage were found to react specifically with TSST-1 but not with other staphylococcal exotoxins. A synthetic peptide (pep3) corresponding to the most frequently identified sequence (phage3) was shown to inhibit binding of all four isolated phage to TSST-1, suggesting that they bind to a common site on TSST-1. Furthermore, pep3 was shown to compete with MHC class II molecules for binding to TSST-1 in a concentration-dependent manner. Comparison of their sequences with MHC class II molecules revealed that phage8 shared sequence homology with two regions of the beta chain of MHC class II molecules: amino acids 57-62, containing a residue (Tyr-60) involved in TSST-1 binding as suggested by X-ray crystallographic data of TSST-1-MHC class II complex; and amino acids 188-193, a region not previously known as a contact domain. These results suggest that the selected sequences recognized the MHC class II binding site on TSST-1. Thus, affinity selection for peptides binding to ligand molecules (e.g., TSST-1) rather than their cognate receptors (e.g., MHC class II) from a random phage display library represents a useful approach to understanding receptor-ligand interactions.

Expression of a bifunctional chimeric protein A-Vargula hilgendorfii luciferase in mammalian cells.

We have designed and constructed a novel chimeric protein that consisted of a single domain of protein A and luciferase derived from sea-firefly Vargula hilgendorfii with the goal of obtaining a heterofunctional immunological tool. The structural gene of luciferase was fused to the 3' terminus of the D domain gene of protein A with/without a short linker of five amino acids. The resulting constructs under the transcriptional regulation of the Rous sarcoma virus (RSV) promoter, were expressed transiently in simian COS-1 and stably in Chinese hamster ovary (CHO) cells. The properties of the resultant chimeric protein were characterized. The results indicated that the dual properties of the chimeric protein could be retained only after the introduction of a linker of (Gly)4 Ser between the two conjugated moieties. Moreover, the chimeric protein was found to retain at least 50% of the specific activity as compared with the non-fused luciferase. The future prospect of the usage of this chimeric protein in the field of diagnostics was further evaluated by performing bioluminescent immunoassays.

Molecular cloning of the immunodominant regions of hepatitis C virus type III and IV by immunoscreening.

We have identified the immunodominant regions of hepatitis C virus (HCV) type III and IV by immunoscreening of a lambda gt11 cDNA library, which was constructed with RNA extracted from pooled sera of patients. In addition to the nucleocapsid protein, nonstructural region 3 (NS3), and NS4 regions, we found that the region at the N terminus of NS5 in type III and IV were also immunoreactive as well as in the prototype and type II, although amino acid homologies were approximately 60% between the prototype (or HCV type II) and HCV isolates of type III or IV. This result indicated that the region at the N terminus of NS5 may be a candidate for serotyping studies of HCV. This report presents the primary structure of immunodominant portions at the N terminus of NS5 in two HCV subtypes and a preliminary study of the immune responses to these antigens.

Intermolecular stacking of gilvocarcin V tetraacetate as evidenced by nuclear magnetic resonance studies.

The temperature-dependent and concentration-dependent 1H NMR studies as well as 13C-relaxation experiments on gilvocarcin V tetraacetate strongly suggest intermolecular stacking of the antibiotic solution.

Revised assignment for the Bacillus subtilis spo0F gene and its homology with spo0A and with two Escherichia coli genes.

The nucleotide sequences of spo0F mutant genes which block the early sporulation process of Bacillus subtilis were determined. The mutation sites together with the results of complementation tests suggested that an open reading frame for a polypeptide of Mr = 14,229 is the spo0F gene. The deduced amino acid sequence shows striking homology with that of the spo0A gene. In addition, the upstream region involving the rib some binding site of the spo0F coding region is also similar to those of spo0A and spo0B. These homologies suggest that all three genes have a similar function in regulating the initiation of sporulation, and that their expression is controlled by a common mechanism. Clear homology is also seen between the spo0 gene products and the transcriptional control proteins, OmpR and Dye, of Escherichia coli suggesting that the spo0 gene products also are involved in the control of transcription.