Split-enzyme fragment as a single affinity tag that enables protein expression,purification, and functional assays

Expressing, isolating, and characterizing recombinant proteins is crucial to many disciplines within the biological sciences. Different molecular tagging technologies have been developed to enable each individual step of protein production, from expression through purification and characterization. Monitoring the entire production process requires multiple tags or molecular interactions, because no individual tag has provided the comprehensive breadth of utility. An ideal molecular tag is small and does not interrupt expression, solubility, folding or function of the protein being purified and can be used throughout the production process. We adapted and integrated a split-luciferase system (NanoBiT®, Promega ®) to perform the range of techniques essential to protein production. We developed a simple method to monitor protein expression in real time to optimize expression conditions. We constructed a novel affinity chromatography system using the split-luciferase system to enable purification. We adapted western blot analysis, enzyme-linked immunosorbent assay, and cell-based bioassay to characterize the expressed proteins. Our results demonstrate that a single-tag can fulfill all aspects needed throughout protein production.     

Denitrification and N2O emissions from a UK pasture soil following the early spring application of cattle slurry and mineral fertiliser

Total denitrification and nitrous oxide (N₂O) losses were measured from three contrasting dairy management systems representing good commercial practice (system 1), production maintained but with reduced N losses (system 2); and nitrate leaching less than 50 mg L^-1 but with reduced production (system 3). Measurements were made following mineral fertiliser application and from two plot experiments where four treatments were applied: control, NH₄NO₃ at 60 kg N ha^-1, cattle slurry applied to the surface (equivalent to 45 kg N ha^-1), and cattle slurry injected. Despite low soil temperatures (<6 °C) and low rainfall (<3 mm), total denitrification and N₂O losses peaked at 56 and 16 g N ha^-1 d^-1, respectively. Total denitrification losses decreased: system 1 system 2 > system 3, whereas N₂O losses decreased: system 2 > system 3 > system 1. Total denitrification losses tended to decrease with decreasing fertiliser application rate, whereas fertiliser application rate was not the sole determinant of the N₂O loss. The system 3 field was injected with cattle slurry for 2 yr, system 2 received some slurry by injection and system 1 received slurry to the surface. Thus, the amount, timing and method of previous cattle slurry application was important in determining the loss following subsequent fertiliser application. For the plot experiments, total denitrification and N₂O losses decreased in the order: slurry injected > mineral fertiliser > slurry applied to the surface > control for 5 days following application. However, 16 and 19 days after application, N₂O losses above the control were measured from plots that had received cattle slurry. It was inferred that the application of cattle slurry to the pasture soil stimulated greater N₂O production and increased losses over a longer time period compared with mineral fertiliser additions.     

Purification,regulation and cloning of a glutathione transferase (GST) from maize resembling the auxin-inducible type-III GSTs

The glutathione transferases (GSTs) from maize (Zea mays L.) with activities toward the chloroacetanilide herbicide metolachlor and the diphenyl ether herbicide fluorodifen were fractionated into two pools based on binding to affinity columns. Pool 1 GSTs were retained on Orange A agarose and were identified as isoenzymes Zea mays (Zm) GST I-I, Zm GST I-II and Zm GST I-III, which have been described previously. Pool 2 GSTs selectively bound to S-hexyl-glutathione-Sepharose and were distinct from the pool 1 GSTs, being composed of a homodimer of 28.5 kDa subunits, termed Zm GST V-V, and a heterodimer of the 28.5 kDa polypeptide and a 27.5 kDa subunit, termed Zm GST V-VI. Using an antibody raised to Zm GST V-VI, a cDNA expression library was screened and a Zm GST V clone identified showing sequence similarity to the type-III auxin-inducible GSTs previously identified in tobacco and other dicotyledenous species. Recombinant Zm GST V-V showed high GST activity towards the diphenyl ether herbicide fluorodifen, detoxified toxic alkenal derivatives and reduced organic hydroperoxides. Antibodies raised to Zm GST I-II and Zm GST V-VI were used to monitor the expression of GST subunits in maize seedlings. Over a 24 h period the Zm GST I subunit was unresponsive to chemical treatment, while expression of Zm GST II was enhanced by auxins, herbicides, the herbicide safener dichlormid and glutathione. The Zm GST V subunit was more selective in its induction, only accumulating significantly in response to dichlormid treatment. During development Zm GST I and Zm GST V were expressed more in roots than in shoots, with Zm GST II expression limited to the roots.     

Forced evolution of a herbicide detoxifying glutathione transferase

Plant Tau class glutathione transferases (GSTUs) detoxify diphenylether herbicides such as fluorodifen, determining their selectivity in crops and weeds. Using reconstructive PCR, a series of mutant GSTUs were generated from in vitro recombination and mutagenesis of the maize sequences ZmGSTU1 and ZmGSTU2 (with the prefix Zm designating Zea mays L.). A screen of 5000 mutant GSTUs identified seven enzymes with enhanced fluorodifen detoxifying activity. The best performing enhanced fluorodifen detoxifying mutant (EFD) had activity 19-fold higher than the parent enzymes, with a single point mutation conferring this enhancement. Further mutagenesis of this residue generated an EFD with a 29-fold higher catalytic efficiency toward fluorodifen as compared with the parents but with unaltered catalysis toward other substrates. When expressed in Arabidopsis thaliana, the optimized EFD, but not the parent enzymes, conferred enhanced tolerance to fluorodifen. Molecular modeling predicts that the serendipitous mutation giving the improvement in detoxification is due to the removal of an unfavorable interaction together with the introduction of a favorable change in conformation of residues 107-119, which contribute to herbicide binding.     

Number of ovarioles and fecundity in the black bean aphid, Aphis fabae

The number of ovarioles in the gonads of the host alternating aphid, Aphis fabae Scop., is not constant. The apterous generations on spindle, emigrants and initial apterous colonists of the secondary host each have a characteristic range of number of ovarioles; subsequent generations in the yearly life cycle have twelve, except for alate exules which have a range and the lowest average number of ovarioles. Ovariole number is programmed and is not a function of adult weight or food quality. The generations with the largest number of ovarioles have the greatest fecundity, highest reproductive rate and smallest offspring, and develop early in the season when the environment is favourable. Later generations have a low fecundity and proportionately more fat, a possible adaptation to the harsher environment these aphids experience.

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Zusammenfassung Die Anzahl der Ovariolen in den Eierstöcken ist bei der wirtswechselnden Bohnenlaus, Aphis fabae, nicht konstant. Bei Läusen der ungeflügelten Generationen auf Pfaffenhütchen, sowie bei Migrantes und den ersten apteren Aphiden auf dem Sekundärwirt variiert die Anzahl der Ovariolen in einem charakteristischen Bereich. Die folgenden Generationen im jährlichen Zyklus haben stets zwölf Ovariolen, ausgenommen alate Exsules, bei denen ebenfalls ein charakteristischer Bereich nachzuweisen war, allerdings mit einem sehr niedrigen Durchschnitt. Die Anzahl der Ovariolen ist programmiert. Ihre Anzahl ist konstant und nicht vom Gewicht der Imagines oder der Nahrungsqualität abhängig. Die Generationen mit der höchsten Anzahl von Ovariolen sind am fruchtbarsten, sie haben die höchste Reproduktionsrate und gebären die kleinsten Läuse. Sie entwickeln sich frühzeitig, wenn die Bedingungen günstig sind. Spätere Generationen zeigen eine niedrigere Fruchtbarkeit, lagern jedoch verhältnismäßig mehr Fett ein, wahrscheinlich eine Anpassung an die schlechteren Bedingungen, denen diese Läuse ausgesetzt sind.

     

Lipid Profile in the Severely Obese: Changes with Weight Loss after Lap‐Band Surgery

Objective: To characterize dyslipidemia before and after weight loss in the severely obese. Research Methods and Procedures: Five hundred fifteen subjects who had Lap‐Band surgery were followed with yearly conventional lipid profiles for up to 4 years. Preoperative data were collected from the most recent 381 subjects, and predictors of dyslipidemia were sought. One hundred seventy‐one subjects completed a 1‐year review, providing data to assess predictors of change in lipids. Results: Favorable changes in fasting triglycerides (TG), high‐density lipoprotein‐cholesterol (HDL‐C), and total cholesterol (TC):HDL‐C ratio occurred within 1 year. All improvements were maintained up to 4 years. Male gender, central obesity, elevated fasting glucose, and insulin resistance were associated with less favorable lipid levels. Fasting plasma glucose best predicted TG (r = 0.46, p < 0.001), whereas insulin sensitivity using the homeostatic model assessment (HOMA %S) correlated best with the HDL‐C (r =0.34, p < 0.001). Higher preoperative fasting glucose best predicted the decrease in TG; improved HOMA %S with weight loss correlated best with HDL‐C. The extent of weight loss had limited influence on lipid changes. However, low preoperative HOMA %S was associated with lower weight loss. Greater weight loss was associated with more favorable lipid measures after controlling for preoperative HOMA %S. Discussion: Dyslipidemia of obesity is related to weight distribution, insulin sensitivity, and impaired glucose tolerance. Improvement with weight loss is related to the decrease in fasting glucose, improvement in insulin sensitivity, and the extent of weight lost. Improvement in dyslipidemia is sustained with long‐term weight loss.     

The effect of etorphine on nicotine- and muscarine-induced catecholamine secretion from perfused rat adrenal glands

The effect of etorphine on nicotine and muscarine-mediated catecholamine (CA) release from isolated perfused rat adrenal glands was investigated. Nicotine increased CA secretion at the low concentration of 0.5 micrograms while higher concentrations of muscarine (5 micrograms) were required. Moreover, muscarine released primarily epinephrine (EP) from rat adrenal glands while nicotine released norepinephrine (NE) and Ep. Etorphine inhibited NE and EP release evoked by nicotine to the same extent, whereas, muscarine-mediated release of NE and EP was not affected. Mecamylamine and verapamil inhibited nicotine but not muscarine-induced CA secretion. Our results suggest that etorphine preferentially interacts with nicotinic receptors on rat adrenal chromaffin cell membranes.