We have used phage display to isolate a range of human domain antibodies (dAbs) that bind to mouse, rat and/or human serum albumin (SA) and can be expressed at very high levels in bacterial, yeast or mammalian cell culture. In contrast to non-SA-binding dAbs, which have terminal half-lives of less than 45 min, the half-lives of these 12 kDa `AlbudAbs' can match the half-life of SA itself. To demonstrate the use of AlbudAbs for extending the half-lives of therapeutic drugs, we created a fusion of the interleukin-1 receptor antagonist (IL-1ra) with an AlbudAb. Soluble IL-1ra is potent inhibitor of IL-1 signalling that is approved for the treatment of rheumatoid arthritis but has a relatively short in vivo half-life. Here we show that although the AlbudAb/IL-1ra fusion has a similar in vitro potency, its in vivo efficacy can be dramatically improved due to its extended serum half-life. AlbudAbs could potentially be used to generate a range of long half-life versions of many different drugs in order to improve their dosing regimen and/or clinical effect.

The manufacture, disposal, and detonation of explosives have resulted in the pollution of large tracts of land and groundwater. Historically, 2,4,6-trinitrotoluene ( TNT) is the most widely used military explosive and is toxic to biological systems and recalcitrant to degradation. To examine the feasibility of enhancing the ability of plants to detoxify the explosive TNT, we created transgenic tobacco ( Nicotiana tabacum) constitutively expressing the nsfI nitroreductase gene from Enterobacter cloacae. The product of TNT reduction by the nitroreductase was found to be 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT). Characterization of the transgenic lines in sterile, aqueous conditions amended with TNT demonstrated that these plants were able to remove all of the TNT from the medium at an initial concentration of 0.5 mM ( 113 mg L-1) TNT. In contrast, growth was suppressed in wild-type plants at 0.1 mM( 23 mg L-1). Following uptake, transgenic seedlings transformed TNT predominantly to 4-HADNT and its high levels appeared to correlate with enhanced tolerance and transformation of TNT. Transformation products of TNT were subsequently conjugated to plant macromolecules to a greater degree in transgenic tobacco, indicating enhanced detoxification compared to the wild type.

An amperometric enzyme electrode was studied based on the wild-type protein trimethylamine dehydrogenase (TMADH), which catalyses the oxidative N-demethylation of trimethylamine to produce dimethylamine and formaldehyde. Ferrocene derivatives were investigated electrochemically, as free diffusing electron acceptors for recycling of the prosthetic groups of the immobilised enzyme. Ferricinium had the highest rates but, inhibited the enzyme, possibly as a result of a conformational change initiated at the Val-344 residue where it binds close to the 4Fe-4S cluster, interrupting the electron transfer between flavin mononucleotide (FMN) and 4Fe-4S by changing the redox potential of one or both of the prosthetic groups. (Dimethylamino)methylene ferrocene (DMAMFe) (k(s) = 0.93 x 10(5) M(-1) s(-1)) did not show inhibition and was used as a comparison for steady-state characterisation. The sensor response was studied over the pH range 6.0-1.0. Plots of kcat/KM revealed two ionisations with pKa values of 7.5 and 10. The pKa of 10 was attributed to the ionisation of the secondary amine in DMAMFe, whereas the pKa of 7.5 was thought to reflect the ionisations of the intramolecular electron pathway. A TMADH/DMAMFe amperometric enzyme electrode was successfully used for the determination of TMA in different fish samples (detection limit: 2 mg TMA-N per lOOg wet fish muscle). The obtained results compared well with a reference method based on picric acid.

Late embryogenesis abundant (LEA) proteins are associated with desiccation tolerance in resurrection plants and in plant seeds, and the recent discovery of a dehydration-induced Group 3 LEA-like gene in the nematode Aphelenchus avenae suggests a similar association in anhydrobiotic animals. Despite their importance, little is known about the structure of Group 3 LEA proteins, although computer modeling and secondary structure algorithms predict a largely alpha-helical monomer that forms coiled coil oligomers. We have therefore investigated the structure of the nematode protein, AavLEA1, in the first such analysis of a well characterized Group 3 LEA-like protein. Immunoblotting and subunit cross-linking experiments demonstrate limited oligomerization of AavLEA1, but analytical ultracentrifugation and gel filtration show that the vast majority of the protein is monomeric. Moreover, CD, fluorescence emission, and Fourier transform-infrared spectroscopy indicate an unstructured conformation for the nematode protein. Therefore, in solution, no evidence was found to support structure predictions; instead, AavLEA1 seems to be natively unfolded with a high degree of hydration and low compactness. Such proteins can, however, be induced to fold into more rigid structures by partner molecules or by altered physiological conditions. Because AavLEA1 is associated with desiccation stress, its Fourier transform-infrared spectrum in the dehydrated state was examined. A dramatic but reversible increase in alpha-helix and, possibly, coiled coil formation was observed on drying, indicating that computer predictions of secondary structure may be correct for the solid state. This unusual finding offers the possibility that structural shifts in Group 3 LEA proteins occur on dehydration, perhaps consistent with their role in anhydrobiosis.

A rational design for the site-specific immobilization of the protein trimethylamine dehydrogenase (TMADH) to facilitate charge transfer between enzyme and an electrode is described. Protein engineering and site-specific chemical modification have been used to extend the electron pathway from the protein surface to redox mediators. The kinetics of TMADH mutants (V344C and Y442C) modified with the iodoacetamide and 4-iodoacetamido 1-naphthole (IAN) showed that modification at position 344 has a more profound influence on intra- and inter-molecular electron transport, and the catalytic parameters k(cat) and K-M(app) became a function of chemical modification. Ferricenium ion was shown to act as an electron acceptor for both mutants, but as its site of interaction is the residue 344, it was rejected for wiring in favour of [Fe(5-NH2-phen)(3)](2+), the latter showing similar very fast homogeneous electron exchange kinetics, ideal for 'wire' construction. The Y442C mutant was successfully immobilised on to an electrode surface which had been chemically modified with the redox polymer, poly-[Fe(5-NH2-phen)(3)](2+). This design enabled direct electrical communication between the enzyme and electrode. Using a partly oxidized polymer to limit the supply of oxidised electron acceptor, gave evidence for transition from the fast "0/2-cycle" to the " 1/3-cycle" for the TMADH.