Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

Background

The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 ~15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A_2AR) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures.

Results

Bioreactor cultures yielded an approximately five times increase in cell density (OD600 ~75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A_2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A_2AR, and therefore more suitable for further functional and structural studies.

Conclusion

Large-scale expression of the A_2AR in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.     

A comparison of Betula pollen seasons at two European sites; Derby, United Kingdom and Poznan, Poland (1995–1999).

A comparison of Betula pollen, animportant European aeroallergen, was undertakenat two sites of similar latitude, Derby, UnitedKingdom and Poznan, Poland from 1995–1999. Bothsites routinely monitor Betula pollenusing a Burkard continuous volumetric sampler.Daily and two-hourly March–June Betulapollen counts per cubic metre of air werestudied at both sites, together withcorresponding meteorological data. Detailedanalysis was undertaken to compare start dates,duration and quantity of Betula pollen.Derby usually had an earlier start of seasonthan Poznan, and both cities showed very littledifference between start dates determined byusing the SUM 75 or 2.5% method. The longestseasonal durations at Derby and Poznan yieldedthe lowest seasonal pollen indexes. Every yearfrom 1995–1999 the Betula seasonal pollenindex was higher in Poznan than in Derby. Poznanhad more daily counts of Betula pollengrains per cubic metre above 500, and at leastone daily count of 1000, each year. At bothsites the yearly seasonal variation correlatedwith the number of April days per year having amaximum temperature of 17 °C or above.There was a similar diurnal periodicity ofApril Betula pollen in Derby and Poznan.Although the two cities, at approximately thesame latitude, have different regional andmeteorological characteristics, the weatherappears to influence Betula pollenseasons in a similar manner. Local clinicianscould use the SUM 75 method as indicative ofthe start of the Betula pollen season atboth sites.     

Quantitative trends in annual totals of five common airborne pollen types (Betula, Quercus, Poaceae, Urtica, and Artemisia), at five pollen-monitoring stations in western Europe

The existence of long-term (20–33 years) trendsin the annual totals of daily airborne pollenconcentrations of five common and/or allergenicwind pollinating taxa was evaluated at fivepollen-monitoring stations in western Europe:Delmenhorst (D), Helmond (NL), Brussels (B),Leiden (NL), and Derby (UK). At all stations,identical or comparable volumetric traps wereused to sample pollen from the air. For grasspollen no increasing or decreasing trends werefound at any station. Trends for birch pollenand oak pollen are increasing, but notsignificant at the stations with the higherannual totals (Delmenhorst and Helmond),probably due to strong year-to-yearfluctuations. At all five stations significantincreasing trends for stinging nettle pollenwere observed. Trends for mugwort pollen aresignificant at all stations, but in differentdirections. Longer periods of observations areneeded to arrive to more definitive conclusionsabout trends in airborne pollenconcentrations.     

A holographic sensor based on a rationally designed synthetic polymer

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. Cross-linking gives the material sufficient spatial integrity to allow a holographic image to be recorded, while maintaining adequate porosity and elasticity of the polymer matrix for sensing applications. The new material has been characterized with respect to its response to pH and compared with a traditional gelatin holographic film. The response to some ions and small molecules typically found in analytical samples has also been measured. Functional groups introduced covalently into the poly(vinyl alcohol) matrix transform the base matrix into a pH-responsive polymer with predictable swelling properties and which can be further derivatized to incorporate specific ligands. A rationally designed holographic sensor for trypsin has been developed from chemically synthesized artificial polymers. A trypsin substrate, the poly(amino acid) poly(L-lysine), was incorporated into poly(vinyl alcohol) holograms to create a 'designed' holographic material which was degraded in a concentration-dependent manner by trypsin. Extensions of this approach to other hydrolytic enzymes are briefly discussed.     

Application of high resolution fast atom bombardment and constant B/E ratio linked scanning to the identification and analysis of acylcarnitines in metabolic disease

Acylcarnitines, a biologically important group of metabolites which have thus far eluded characterization by mass spectrometry, exhibit very intense fast atom bombardment mass spectra dominated by parent cations. The combination of high resolution selected ion detection and daughter ion analysis using the linked scan at constant B/E ratio has enabled the unequivocal identification of the acylcarnitines in the urine of children with propionic acidemia, methylmalonic aciduria and Reye's Syndrome. Quantitative analysis of acetylcarnitine and propionylcarnitine in selected samples was accomplished by isotope dilution, utilizing (2H3)acetyl- and (2H5)propionylcarnitines as internal standards. These techniques will allow assessment of the therapeutic use of L-carnitine in these disorders.     

A study of Gramineae and Urticaceae pollen in the Derby area

Summary Gramineae and Urticaceae seasonal variations and diurnal rhythms for Derby in 1986, 1987 and 1988 are shown and discussed.The time of highest hourly concentration of Gramineae for Derby in June was 1900 hours. Maximum hourly counts for June of Urticaceae pollen occurred between 1400 and 1600 hours.Urticaceae has a second period of high concentration in August. Gramineae and Urticaceae pollens from 1969–1990 show no significant long term trends.     

Gonadal steroid and chronic morphine treatment do not change the posttranslational processing of beta-endorphin in the rat brain

The present study examines whether two treatments known to induce refractoriness to exogenous morphine produce this desensitization through a change in the posttranslational processing of brain beta-endorphin (beta-End). The first experiment examined whether an ovarian steroid regimen which produces a transient desensitization of brain opiate receptor mechanisms alters beta-End processing in the preoptic area (POA), medial basal hypothalamus (MBH), and brainstem (BS). The second experiment monitored the effects of morphine pellet treatment, known to produce morphine dependency, on immunoreactive beta-End forms in the hypothalamus and periaquaductal gray area of the midbrain (PAG). The individual molecular forms of beta-End were separated using ion exchange chromatography and collection fractions were quantitated for beta-End immunoreactivity by RIA. The results show that regional differences occur in the posttranslational processing of beta-End. In the hypothalamus, MBH and POA, beta-End-(1-31) and its non-acetylated C-terminal cleavage products, beta-End-(1-27) and beta-End-(1-16) were the predominant forms of beta-End. The PAG pools produced a beta-End peptide elution profile similar to the hypothalamus, although small amounts of N-acetyl-beta-End-(1-31) were also identified. The BS exhibited the least posttranslational processing of beta-End; beta-End-(1-31) was the primary product with smaller amounts of beta-End-(1-27) and beta-End-(1-26) observed. However, neither ovarian steroid treatment nor chronic morphine produced any changes in posttranslational processing of beta-End or in total beta-End concentration in any of the brain regions examined in these experiments. These data indicate that the refractoriness or tolerance to exogenous morphine associated with steroid or chronic morphine treatment cannot be explained by alterations in the biological activity of beta-End resulting from the differential regulation of its posttranslational processing products.     

Rapid optimization of enzyme substrates using defined substrate mixtures.

A strategy is described for the rapid optimization of kcat/Km for protease substrates. Selected positions of a given peptide substrate sequence are varied through synthesis with mixtures of amino acids. Incubation of the resulting peptide mixture with the enzyme of interest and analysis by high pressure liquid chromatography provides a direct measure of analogs with enhanced kcat/Km. High performance liquid chromatography/continuous flow fast atom bombardment mass spectrometry is used to assign structure to each peak in the chromatogram. As an example of the utility and efficiency of "substrate mapping" we describe optimization of the collagenase substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 (where Dnp is dinitrophenyl) at the P'1 and P'2 positions. Six different mixtures were prepared for evaluation, representing the synthesis of 128 different synthetic substrates. "Substrate mapping" has led to Dnp-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-NH2, a substrate that possesses a 10-fold better kcat/Km than Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2.