Production of bifunctional proteins by Aspergillus awamori: llama variable heavy chain antibody fragment (V(HH)) R9 coupled to Arthromyces ramosus peroxidase (ARP)

The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment V_HH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which produced up to 30 mg l⁻¹ fusion protein in the culture medium. Both fusion proteins showed peroxidase activity in an ABTS activity test. Considerable amounts of fusion protein were detected intracellularly, suggesting that the fungus encounters problems in secreting these kind of proteins. ELISA experiments showed that ARP-R9 was less able to bind its antigen, the azo-dye RR6, as compared to R9-ARP. Furthermore, in contrast to R9-ARP, ARP-R9 bound to RR6 did not show peroxidase activity anymore. These results indicate that fusion of ARP to the C-terminus of the antibody fragment V_HH R9 (R9-ARP) is the preferred orientation.     

A new approach to directed gene evolution by recombined extension on truncated templates (RETT)

We describe a new approach to in vitro DNA recombination technique termed recombined extension on truncated templates (RETT). RETT generates random recombinant gene library by template-switching of unidirectionally growing polynucleotides from primers in the presence of unidirectional single-stranded DNA fragments used as templates. RETT was applied to the recombination of two homologous chitinase genes from S. marcescens ATCC 21074 and S. liquefaciens GM1403. When the shuffled genes were examined by restriction mapping and sequence analysis, it was found that chimeric genes were produced at a high frequency (more than 70%) between two chitinase genes with 83% of sequence identity. The number of crossovers within each chimeric gene ranged from one to four, and the recombination points were randomly distributed along entire DNA sequence. We also applied RETT to directed evolution of a chitinase variant for enhancing thermostability. Chimeric chitinases that were more thermostable than the parental enzyme were successfully obtained by RETT-based recombination.     

Ambient temperature limits and stability of temperature regulation in telemetered male and female rats

Knowing the ambient temperature (T_a) limits of normothermia in laboratory rodents is important because their thermoregulatory responses are useful in studies of physiology, pharmacology and toxicology. The present study assessed the T_a limits of normothermia using radiotelemetry to monitor core temperature (T_c), heart rate (HR), and motor activity (MA) in unrestrained, male and female Long-Evans rats over a 24 h period. Rats were housed individually in acrylic cages with wire-screen tops and bottoms and maintained at T'_as ranging from 12 to 33.5°C for 24 h with food and water provided ad libitum on a 12:12 L:D photoperiod. The limits of normothermia (i.e. where there was no significant change in T_c) were <12–29.5°C for females and 14.5–29.5°C for males. T_c of males at T'_as of 12, 32, and 33.5°C increased significantly above the baseline T_c. Female rats had a lower T_c than males at the warmest and coldest T'_as. HR and MA were generally higher in females at all T'_as. Males appeared to be poorly adapted to thermoregulate at T'_as above 30°C as based on their excessively high T'_cs, low MA, and marked weight loss compared to that of the females. Within the limits of normothermia the stability of T_c regulation (i.e. [(ΔT_c/ΔT_a) × 100]/2) was ±1.3 and ± 0.9% for males and females, respectively, over a 24 h period. These data on the stability of T_c in the male and female rat provide a valuable framework to study the acute and chronic effects of drugs, chemicals and other agents that affect temperature regulation.     

Extending the diversity of cytochrome P450 enzymes by DNA family shuffling

The cytochrome P450 enzymes involved in xenobiotic metabolism are an excellent starting point for the directed evolution of novel biocatalysts due to their wide substrate specificity. A shuffled library of three highly homologous mammalian genes (for P450 2C9, P450 2C11 and P450 2C19) was constructed by applying a modified DNA family shuffling procedure. The modifications made to the traditional DNA shuffling protocols involved non-random digestion via the use of different combinations of restriction enzymes (REs) followed by isolation of fragments under 300 bp by size-selective filtration. Shuffled cytochrome P450 mutants were co-expressed in Escherichia coli with their redox partner, NADPH-cytochrome P450 reductase (NPR). We report here how non-random fragmentation may help in chimeragenesis within the areas of low sequence similarity such as substrate recognition sites (SRSs) that are generally underrepresented in recombination using the random fragmentation process. Size-selective filtration was used to limit recovery of incompletely digested fragments and consequently minimize the chances for contamination of the shuffled library with parental forms. No parental forms could be detected in the shuffled library using restriction fragment length polymorphism (RFLP) analysis, suggesting the library was free of parental contamination. Sequencing of randomly selected mutants demonstrated a high level of chimeragenesis with on average of 8.0+/-2.2 crossovers and a low level of mutagenesis with 5.2+/-2.8 spontaneous mutations per approximately 1.5 kbp of the full-length P450 sequence. The proportion of properly folded protein as indicated by the observation of characteristic Fe(II).CO vs. Fe(II) difference spectra was 15% (4/27) of analysed mutants. Screening of the shuffled library for indole oxidation revealed four clones with similar or higher levels of indigo pigment production to those of the parental P450s and two clones with elevated P450 expression. In this paper we present a method for the effective family shuffling of cytochrome P450 enzymes, applicable to the creation of mutant libraries with expanded metabolic diversity and with a significant proportion of functional clones.     

Virulence of entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae) against Tipula paludosa (Diptera: Tipulidae), a turfgrass pest on golf courses

The European crane fly (ECF), Tipula paludosa Meigen feeds on leaves, crowns, and roots of cool-season turfgrasses causing damage to residential lawns and golf courses. A laboratory study was conducted to determine the susceptibility of ECF larvae to four commercial entomopathogenic nematode (EPN) species (Heterorhabditis marelatus, H. megidis, Steinernema carpocapsae and S. feltiae). The virulence of four S. feltiae isolates recovered from golf courses in Quebec and Ontario were also compared to a commercial strain. LC₅₀ values of EPN against late instar ECF larvae were 152, 562, 763, and 3584 for S. feltiae, H. megidis, H. marelatus and S. carpocapsae, respectively. When non-feeding (without grass seedling), ECF larvae mortalities decreased for all nematode species and concentrations tested. At 25°C, LC₅₀ values for the two most virulent indigenous S. feltiae were 129 and 187 nematodes/larva, not different from the commercial strain. At 5°C, the commercial S. feltiae was more effective than both BIC14A and RE6A isolates against ECF larvae. However, at 15°C, BIC14A was the most virulent at the low concentration of 200 IJs/larva.     

Hypoallergens for allergen-specific immunotherapy by directed molecular evolution of mite group 2 allergens

Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wild-type allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.     

Thermoregulation by the short-horned lizard (Phrynosoma douglassi) at high elevation

The short-horned lizard Phrynosoma douglassi was studied in a montane habitat (elevation of 2290 m) in the Guadalupe Mountains of Texas. The body temperatures (T_b) of active lizards were consistently between 35–36°C (grand mean=35.5°C) in the period from 0900 to 1800 h during the months of May through September. The lizards began their spring activity during April when environmental temperatures were still low. Although T_bs in April were significantly lower than during May through September, on sunny April days the lizards were able to maintain T_bs near 30°C even when air temperatures were as low as 1.5°C. P. douglassi at this site thermoregulated very effectively whenever they were not limited by the physical environment.     

Comparative thermodynamic elucidation of the structural stability of thermophilic proteins

Differential scanning calorimetry, circular dichroism, and visible absorption spectrophotometry were employed to elucidate the structural stability of thermophilic phycocyanin derived from Cyanidium caldarium, a eucaryotic organism which contains a nucleus, grown in acidic conditions (pH 3.4) at 54°C. The obtained results were compared with those previously reported for thermophilic phycocyanin derived from Synechococcus lividus, a procaryote containing no organized nucleus, grown in alkaline conditions (pH 8.5) at 52°C. The temperature of thermal unfolding (t_d) was found to be comparable between C. caldarium (73°C) and S. lividus (74°C) phycocyanins. The apparent free energy of unfolding (ΔG_[urea]=0) at zero denaturant (urea) concentration was also comparable: 9.1 and 8.7 kcal/mole for unfolding the chromophore part of the protein, and 5.0 and 4.3 kcal/mole for unfolding the apoprotein part of the protein, respectively. These values of t_d and ΔG_[urea]=0 were significantly higher than those previously reported for mesophilic Phormidium luridum phycocyanin (grown at 25°C). These findings revealed that relatively higher values of t_d and ΔG_[urea]=0 were characteristics of thermophilic proteins. In contrast, the enthalpies of completed unfolding (ΔH_d) and the half-completed unfolding (ΔH_d)1/2 for C. caldarium phycocyanin were much lower than those for S. lividus protein (89 versus 180 kcal/mole and 62 versus 115 kcal/mole, respectively). Factors contributing to a lower ΔH_d in C. caldarium protein and the role of charged groups in enhancing the stability of thermophilic proteins were discusse.     

Genome shuffling of Streptomyces viridochromogenes for improved production of avilamycin

Avilamycin is one of EU-approved antimicrobial agents in feed industry to inhibit the growth of multidrug-resistant Gram-positive bacteria. Here, we applied a process of combining ribosome engineering and genome shuffling to achieve rapid improvement of avilamycin production in Streptomyces viridochromogenes AS 4.126. The starting mutant population was generated by ⁶⁰Co γ-irradiation treatments of the spores. After five rounds of protoplast fusion with streptomycin-resistance screening, an improved recombinant E-219 was obtained and its yield of avilamycin reached 1.4 g/L, which was increased by 4.85-fold and 36.8-fold in comparison with that of the shuffling starter Co γ-316 and the ancestor AS 4.126. Furthermore, the mechanism for the improvement of shuffled strains was investigated. Recombinants with enhanced streptomycin resistance exhibited significantly higher avilamycin production and product resistance, probably due to the mutations in the ribosome protein S12. The morphological difference between the parent mutant and shuffled recombinant was observed in conidiospore, and hyphae pellets. The presence of genetic diversity among shuffled populations with varied avilamycin productivity was confirmed by randomly amplified polymorphic DNA analysis. In summary, our results demonstrated that genome shuffling combined with ribosome engineering was a powerful approach for molecular breeding of high-yield industrial strains.     

Schistosoma mansoni and Schistosoma japonicum: Utilization of amino acids

, , and 1972. Schistosoma mansoni and Schistosoma japonicum: utilization of amino acids. International Journal for Parasitology 2: 425–430. The production of ¹⁴CO₂ from 12 labeled amino acids by S. mansoni and S. japonicum was studied. No ¹⁴CO₂ was detected from incubations with glycine, isoleucine, leucine, lysine or phenylalanine. Differences were found between sexes and/or species for the other amino acids studied. Species related differences included a greater rate of metabolism of glutamic and aspartic acid by S. mansoni than by S. japonicum. Proline and histidine were utilized by S. mansoni males and females, respectively. S. japonicum male worms did not utilize proline, while histidine was not utilized by the female of this species. Major sex related differences included greater ¹⁴CO₂ production from glutamic acid, aspartic acid and arginine by S. mansoni males than by females, and the utilization of histidine by male S. japonicum but not by females. Incubation in tyrosine resulted in the release of only small amounts of ¹⁴CO₂ by female worms of both species but no ¹⁴CO₂ production by male worms.