Incremental truncation as a strategy in the engineering of novel biocatalysts

The application and success of combinatorial approaches to protein engineering problems have increased dramatically. However, current directed evolution strategies lack a combinatorial methodology for creating libraries of hybrid enzymes which lack high homology or for creating libraries of highly homologous genes with fusions at regions of non-identity. To create such hybrid enzyme libraries, we have developed a series of combinatorial approaches that utilize the incremental truncation of genes, gene fragments or gene libraries. For incremental truncation, Exonuclease III is used to create a library of all possible single base-pair deletions of a given piece of DNA. Incremental truncation libraries (ITLs) have applications in protein engineering as well as protein folding, enzyme evolution, and the chemical synthesis of proteins. In addition, we are developing a methodology of DNA shuffling which is independent of DNA sequence homology.     

A rolling-gliding wear simulator for the investigation of tribological material pairings for application in total knee arthroplasty

Background  

Material wear testing is an important technique in the development and evaluation of materials for use in implant for total knee arthroplasty. Since a knee joint induces a complex rolling-gliding movement, standardised material wear testing devices such as Pin-on-Disc or Ring-on-Disc testers are suitable to only a limited extent because they generate pure gliding motion only.     

As easy as flipping a switch?

Proteins that behave as switches help to establish the complex molecular logic that is central to biological systems. Aspiring to be nature's equal, researchers have successfully created protein switches of their own design; in particular, numerous and varied zinc-triggered switches have been made. Recent studies in which such switches have been readily identified from combinatorial protein libraries support the notion that proteins are primed to show allosteric behavior and that newly created ligand-binding sites will often be functionally coupled to the original activity of the protein. If true, this notion suggests that switch engineering might be more tractable than previously thought, boding well for the basic science, sensing and biomedical applications for which protein switches hold much promise.     

Geographical variation in temporal and spatial vocalization patterns of male harbour seals in the mating season

In the aquatically mating harbour seal, Phoca vitulina, oestrous females show marked differences in spatial and temporal distribution between geographical areas. This suggests that the males' display behaviour may also vary between areas. We recorded male vocalizations in two areas, the Moray Firth and Orkney, U.K. In the Moray Firth, females haul out on a few intertidal sandbars and travel along predictable routes to forage at sea. In Orkney, female haul out sites are much less influenced by tidal availability and females are much more dispersed. In the Moray Firth, males vocalized only during a short mating season, from 1 July to 12 August. Vocalizations varied significantly with the tide, the peak at high tide clearly coinciding with the period when most females were in the water. In contrast, vocalizations in Orkney were significantly related to both tidal and diel patterns. We suggest that the timing of male vocalizations reflects differences in female availability between sites. In the inner Moray Firth, vocalizations were heard throughout the females' range, whereas vocalizations in Orkney were heard only in two discrete areas. However, at both sites the density of vocalizing males was highest in narrow channels and/or along predictable female travel routes. Therefore, males clearly adapt their temporal and spatial behaviour patterns to variations in female distribution and density. These results suggest that male mating strategies in aquatically mating pinnipeds are more variable than was previously envisaged. Copyright 1999 The Association for the Study of Animal Behaviour.     

Global functional profiling of gene expression

The typical result of a microarray experiment is a list of tens or hundreds of genes found to be differentially regulated in the condition under study. Independent of the methods used to select these genes, the common task faced by any researcher is to translate these lists of genes into a better understanding of the biological phenomena involved. Currently, this is done through a tedious combination of searches through the literature and a number of public databases. We developed Onto-Express (OE) as a novel tool able to automatically translate such lists of differentially regulated genes into functional profiles characterizing the impact of the condition studied. OE constructs functional profiles (using Gene Ontology terms) for the following categories: biochemical function, biological process, cellular role, cellular component, molecular function, and chromosome location. Statistical significance values are calculated for each category. We demonstrate the validity and the utility of this comprehensive global analysis of gene function by analyzing two breast cancer datasets from two separate laboratories. OE was able to identify correctly all biological processes postulated by the original authors, as well as discover novel relevant mechanisms.     

Creation of an allosteric enzyme by domain insertion

Two allosteric enzymes have been created by the covalent linkage of non-interacting, monomeric proteins with the prerequisite effector-binding and catalytic functionalities, respectively. This was achieved through a combinatorial process called random domain insertion. The fragment of the TEM-1 beta-lactamase gene coding for the mature protein lacking its signal sequence was randomly inserted into the Escherichia coli maltose-binding protein (MBP) gene to create a domain insertion library. This library's diversity derived both from the site of insertion and from a distribution of tandem duplications or deletions of a portion of the MBP gene at the insertion site. From a library of approximately 2 x 10(4) in-frame fusions, approximately 800 library members conferred a phenotype to E.coli cells that was consistent with the presence of bifunctional fusions that could hydrolyze ampicillin and transport maltose in E.coli. Partial screening of this bifunctional sublibrary resulted in the identification of two enzymes in which the presence of maltose modulated the rate of nitrocefin hydrolysis. For one of these enzymes, the presence of maltose increased k(cat) by 70% and k(cat)/K(m) by 80% and resulted in kinetic parameters that were almost identical to TEM-1 beta-lactamase. Such an increase in activity was only observed with maltooligosaccharides whose binding to MBP is known to induce a conformational change. Modulation of the rate of nitrocefin hydrolysis could be detected at maltose concentrations less than 1 microM. Intrinsic protein fluorescence studies were consistent with a conformational change being responsible for the modulation of activity.     

Enhanced catalytic efficiency of aminoglycoside phosphotransferase (3')-IIa achieved through protein fragmentation and reassembly

Many monomeric proteins can be split into two fragments, yet the two fragments can associate to make an active heterodimer. However, for most locations in a protein such a conversion is not feasible, presumably due to inefficient assembly or improper folding of the fragments. For some locations, this can be overcome by fusion of the fragments to dimerization domains that facilitate correct assembly. A variety of heterodimers of aminoglycoside phosphotransferase (3')-IIa (Neo) were created in which the Neo fragments required fusion to a pair of leucine zippers for activity in vivo. However, the ability of these heterodimers to confer kanamycin resistance to Escherichia coli cells was impaired compared to wild-type Neo, primarily due to poor production of soluble protein. The mutations R177S and V198E restored the kanamycin resistance to wild-type levels while maintaining the dependence on leucine zippers for activity. These mutations restored high levels of kanamycin resistance not through an improvement in the production of soluble protein but rather by conferring a large improvement in k(cat)/K(m), surpassing that of Neo. Furthermore, whereas R177S and V198E served to improve k(cat)/K(m) 60-fold in the context of the heterodimer, the same mutations in the context of wild-type Neo had a ninefold negative effect on k(cat)/K(m). This demonstrates the possibility that enzymes with improved catalytic properties can be created through a process involving fragmentation and fusion to domains that facilitate assembly of the fragments.     

A limited survey of aflatoxins and fumonisins in retail maizebased products in the UK using immunoassay detection

A total of 27 maize-based products destined for human consumption were collected from retail outlets within the city of Glasgow in the UK and were analysed for the presence of aflatoxins using immunoaftinity column chromatography with fluorescence detection and for fumonisins by competitive ELISA. Aflatoxins were detected at a trace level below 4 in eight (30%) of the 27 samples tested, no sample contained aflatoxins at a high level although one sample of sweetcorn did contain aflatoxins at a level of 5-10 Fumonisins were detected in eight (30%) of the samples at levels from 1 to 8mgkg^-1 and a further eight samples contained fumonisin at a level below 1 mgkg^-1 but above the detectable level. The highest concentration of fumonisins was found in a sample of fine corn meal at 8-12mgkg^-1.     

Intensive nitrogen loss over the Omani Shelf due to anammox coupled with dissimilatory nitrite reduction to ammonium

A combination of stable isotopes (¹⁵N) and molecular ecological approaches was used to investigate the vertical distribution and mechanisms of biological N₂ production along a transect from the Omani coast to the central–northeastern (NE) Arabian Sea. The Arabian Sea harbors the thickest oxygen minimum zone (OMZ) in the world''s oceans, and is considered to be a major site of oceanic nitrogen (N) loss. Short (<48 h) anoxic incubations with ¹⁵N-labeled substrates and functional gene expression analyses showed that the anammox process was highly active, whereas denitrification was hardly detectable in the OMZ over the Omani shelf at least at the time of our sampling. Anammox was coupled with dissimilatory nitrite reduction to ammonium (DNRA), resulting in the production of double-¹⁵N-labeled N₂ from ¹⁵NO₂⁻, a signal often taken as the lone evidence for denitrification in the past. Although the central–NE Arabian Sea has conventionally been regarded as the primary N-loss region, low potential N-loss rates at sporadic depths were detected at best. N-loss activities in this region likely experience high spatiotemporal variabilities as linked to the availability of organic matter. Our finding of greater N-loss associated with the more productive Omani upwelling region is consistent with results from other major OMZs. The close reliance of anammox on DNRA also highlights the need to take into account the effects of coupling N-transformations on oceanic N-loss and subsequent N-balance estimates.