Judy Halliday

Molecular diversity through sugar scaffolds.

Monosaccharides provide an excellent platform to tailor molecular diversity by appending desired substituents at selected positions around the sugar scaffold. The presence of five functionalized and stereo-controlled centres on the sugar scaffolds gives the chemist plenty of scope to custom design molecules to a pharmacophore model. This review focuses on the peptidomimetic developments in this area, as well as the concept of tailoring structural and functional diversity in a library using carbohydrate scaffolds and how this can lead to increased hit rates and rapid identification of leads, which has promising prospects for drug development.

The complete amino acid sequence of feline β-lactoglobulin II and a partial revision of the equine β-lactoglobulin II sequence

Abstract The amino acid sequence of feline β-lactoglobulin (designated II) has been determined. The protein chain is 163 amino acids long with a relative molecular mass of 18558. The primary structure was determined by sequencing of native protein (residues 1–25), BPNS-skatole cleavage fragments and the peptides obtained by proteolytic cleavage with V8 proteinase and TPCK-trypsin. Feline β-lactoglobulin II has 53 and 57% positional identities with bovine β-lactoglobulin A and equine β-lactoglobulin I, respectively, and approx. 68% with a revised sequence of equine β-lactoglobulin II. The equine β-lactoglobulin II sequence was re-examined between positions 78 and 122 resulting in a major revision in this area with only a single insertion to give a total of 163 residues.

Crystallization and preliminary diffraction studies of native and selenomethionine CcmG (CycY, DsbE)

Disulfide-bond (Dsb) proteins are a family of redox proteins containing a Cys-X-X-Cys motif. They are essential for disulfide-bond exchange in the bacterial periplasm and are necessary for the correct folding and function of many secreted proteins. CcmG (DsbE) is a reducing Dsb protein required for cytochrome c maturation. Crystals of Bradyrhizobium japonicum CcmG have been obtained that diffract X-rays to 1.14 A resolution. The crystals are orthorhombic, space group P2(1)2(1)2(1), with unit-cell parameters a = 35.1, b = 48.2, c = 90.2 A. Selenomethionine CcmG was expressed without using a methionine auxotroph or methionine-pathway inhibition and was purified without reducing agents.

Biological Diversity from a Structurally Diverse Library: Systematically Scanning Conformational Space Using a Pyranose Scaffold†

Success in discovering bioactive peptide mimetics is often limited by the difficulties in correctly transposing known binding elements of the active peptide onto a small and metabolically more stable scaffold while maintaining bioactivity. Here we describe a scanning approach using a library of pyranose-based peptidomimetics that is structurally diverse in a systematic manner, designed to cover all possible conformations of tripeptide motifs containing two aromatic groups and one positive charge. Structural diversity was achieved by efficient selection of various chemoforms, characterized by a choice of pyranose scaffold of defined chirality and substitution pattern. A systematic scanning library of 490 compounds was thus designed, produced, and screened in vitro for activity at the somatostatin (sst(1-5)) and melanin-concentrating hormone (MCH(1)) receptors. Bioactive compounds were found for each target, with specific chemoform preferences identified in each case, which can be used to guide follow-on drug discovery projects without the need for scaffold hopping.

Feline whey proteins: Identification, isolation and initial characterization of α-lactalbumin, β-lactoglobulin and lysozyme

Abstract 1. 1. Both α-lactalbumin and β-lactoglobulin-like proteins were detected in the whey fraction of feline milk by immunoblotting with rabbit antisera to α-lactalbumin and β-lactoglobulin, respectively. 2. 2. α-Lactalbumin was found to occur in both glycosylated and unglycosylated forms in approximately equal concentrations. No polymorphism of feline α-lactalbumin was found. 3. 3. Feline β-lactoglobulin-like proteins produced complex electrophoretic pattenrs that appears to be determined by three distinct loci. Between two and five genetic variants are expressed by each locus. 4. 4. Lysozyme was detected at levels of approximately 1 mg/ml in skim milk. 5. 5. The identification of the proteins as α-lactalbumin, β-lactoglobulin and lysozyme were confirmed by determination of N- terminal amino acid sequences.

Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1 families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c--both tagged and detagged--from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies.

Characterization of Baboon (Papio hamadryas) Milk Proteins

The major proteins of baboon milk were identified as β-lactoglobulin (βLG), α-lactalbumin (αLA), lysozyme, lactoferrin, casein, and albumin by immobiline isoelectric focusing, SDS-PAGE, immunoblotting of gels with rabbit antisera to human αLA, lysozyme, and albumin and bovine βLG and casein, and N-terminal sequencing of proteins blotted from gels. The first 30 N-terminal residues of baboon βLG are identical to those of macaque (Macaca fasicularis) βLG except for a (D/N) polymorphism at residue 2. The complete cDNA sequence and derived amino acid composition of βLG were elucidated using RT-PCR amplification of poly(A)+ mRNA purified from lactating mammary gland. Baboon βLG consists of 168 amino acid residues (Mr 20,750) and is the longest βLG identified to date. βLG and αLA polymorphisms with three (A, B, and C) and two (A and B) variants, respectively, were detected by immobiline IEF, pH 4–6, of individual baboon milk samples at varying stages of lactation.

Feline and canine milk lysozymes.

1. The electrophoretic characteristics of feline (Felis catus) and canine (Canis familiaris) milk lysozymes were studied using starch gel electrophoresis and isoelectric focusing. 2. Feline milk lysozyme was found to be polymorphic (two variants, designated A and B with frequencies of 0.13 and 0.87, respectively). Canine milk lysozyme was not polymorphic. 3. The lytic activities of feline and canine milk lysozymes were examined in buffers of varying pH and ionic strength. Preliminary kinetics studies were done. 4. Maximal lytic activity for both lysozymes was found in imidazole-HCl buffer pH 7.4 and both exhibited second order reaction kinetics. 5. Amino acid compositions of both lysozymes were determined.

Commercial Aspects of Vaccine Development

The development of any therapeutic for human or animal use is a lengthy and highly regulated process. There are multiple factors that are important in assessing commercial attractiveness in the development of any new vaccine, and continual evaluation of these factors during the process is essential in developing new and improved vaccines. This chapter reviews the challenges associated with vaccine development and commercialization generally in the context of the various classes of micro- and nanotechnology vaccines and vaccine delivery devices that are in clinical development. The commercial challenges as well as potential advantages associated with the development of nanotechnology vaccines will be discussed.