Journal articles

A combination of rational design based on mimicking natural protein-carbohydrate interactions and solid-phase combinatorial chemistry has led to the identification of an affinity ligand which displays selectivity for the mannose moiety of glycoproteins. The ligand, denoted 18/18 and comprising a triazine scaffold bis-substituted with 5-aminoindan, has been synthesised in solution, characterised by TLC, 1H-NMR and MS. When immobilised to amine-derivatised agarose at concentrations >24 micromol/g moist weight gel, ligand 18/18 selectively binds glucose oxidase.

The structural gene, adhA, for a thermostable primary alcohol dehydrogenase was cloned from Thermoanaerobacter ethanolicus JW200. Constitutive expression from its own promoter was observed in Escherichia coli. The nucleotide sequence of adhA corresponded to an open reading frame of 1197 bp, encoding a polypeptide of 399 amino acids with a calculated Mr of 43 192. Amino acid sequence analysis showed 67-69% identity with alcohol dehydrogenases from two archaeal species and 29-37% identity with bacterial type III alcohol dehydrogenases.

Biology can teach the physical world of electronics, computing, materials science and manufacturing how to assemble complex functional devices and systems that operate at the molecular level. Our present capability to fabricate simple molecular tools, devices, materials and machines is primitive compared with the sophistication of nature. Nevertheless, the nanomanufacturing of 'biomimetic' devices is moving ahead strongly.

A new approach for the identification of ligands for the purification of pharmaceutical proteins by affinity chromatography is described. The technique involves four steps. Selection of an appropriate site on the target protein, design of a complementary ligand compatible with the three-dimensional structure of the site, construction of a limited solid-phase combinatorial library of near-neighbour ligands and solution synthesis of the hit ligand, immobilisation, optimisation and application of the adsorbent for the purification of the target protein.

A synthetic bifunctional ligand (22/8) comprising a triazine scaffold substituted with 3-aminophenol (22) and 4-amino-1-naphthol (8) has been designed, synthesised, characterised and immobilized on agarose beads to create a robust, highly selective affinity adsorbent for human immunoglobulin G (IgG). Scatchard analysis of the binding isotherm for IgG on immobilized 22/8 (90 micromol 22/8/g moist weight gel) indicated an affinity constant (Ka) of 1.4 x 10(5) M(-1) and a theoretical maximum capacity of 151.9 mg IgG/g moist weight gel.

This paper describes the construction of a sensor for the direct monitoring of a recombinant protein, the human insulin analogue (MI3). The surface plasmon resonance (SPR) sensor incorporates an immobilised, sterilisable affinity-ligand that has been designed to bind to MI3.

An IgG-binding ligand library comprising 88 adsorbents based on a known lead compound (Li et al., 1998) was generated on an agarose solid phase. Individual members of the library were synthesized in two chemical steps using cyanuric chloride as the scaffold immobilized on the beaded support. The library was screened for binding of pure human IgG, whence selected ligands from the library were further assessed for specificity by the purification of IgG from human plasma.

The concepts of rational design and solid phase combinatorial chemistry were used to develop affinity adsorbents for glycoproteins. A detailed assessment of protein-carbohydrate interactions was used to identify key residues that determine monosaccharide specificity, which were subsequently exploited as the basis for the synthesis of a library of glycoprotein binding ligands. The ligands were synthesised using solid phase combinatorial chemistry and were assessed for their sugar-binding ability with the glycoenzymes, glucose oxidase and RNase B.

A new silver halide-containing holographic recording material has been designed and developed specifically for holographic chemical sensors. The hologram enables very small volume changes to be measured in a polymer layer throughout which the hologram is located. The holographic film is based on a fine-grain silver bromide emulsion suspended in a poly(vinyl alcohol) matrix crosslinked with Cr(III) ions.

New opportunities for biosensors are now appearing in clinical and genetic diagnostics, genomics, environmental protection, food processing and safety, drug discovery and bioprocess monitoring. Concerns about the cost, stability and selectivity of previous sensor technologies are being addressed by developing new recognition systems and their integration into transducers, micro- and nanofabricated devices, array technologies and novel magnetic, acoustic and optical transduction systems.