Optimised fermentation strategy for 13C/15N recombinant protein labelling in Escherichia coli for NMR-structure analysis.

A widely applicable cultivation strategy, which reduces the costs of expensive isotopes, is designed for maximal (98-100%) incorporation of [13C] and [15N] into labelled recombinant protein expressed in Escherichia coli, allowing better assignment of the resonances for NMR studies. Isotope labelling of the culture was performed throughout the complete process, starting from preculture. Sufficient biomass is first generated in a batch phase. Upon consumption of glucose, identified by a sharp drop of on-line monitored oxygen consumption, expression is induced and cultivation is continued under glucose-limited conditions as fed-batch process. Thereby a quantitative utilisation of the most expensive component [13C]-glucose is achieved, while the approximate amount of the [15N]-ammonium chloride to be incorporated is calculated from the scheduled biomass. The usefulness of the strategy is demonstrated with production of uniformly [13C/15N]-labelled tryparedoxin of Crithidia fasciculata. Ideal isotope incorporation and product quality is documented by MALDI-TOF mass spectrometry and two- and three-dimensional NMR spectra.

Structures of tryparedoxins revealing interaction with trypanothione.

Tryparedoxins (TXNs) catalyse the reduction of peroxiredoxin-type peroxidases by the bis-glutathionyl derivative of spermidine, trypanothione, and are relevant to hydroperoxide detoxification and virulence of trypanosomes. The 3D-structures of the following tryparedoxins are presented: authentic tryparedoxin1 of Crithidia fasciculata, CfTXN1; the his-tagged recombinant protein, CfTXN1H6; reduced and oxidised CfTXN2, and an alternative substrate derivative of the mutein CfTXN2H6-Cys44Ser. Cys41 (Cys40 in TXN1) of the active site motif 40-WCPPCR-45 proved to be the only solvent-exposed redox active residue in CfTXN2. In reduced TXNs, its nucleophilicity is increased by a network of hydrogen bonds. In oxidised TXNs it can be attacked by the thiol of the 1N-glutathionyl residue of trypanothione, as evidenced by the structure of 1N-glutathionylspermidine-derivatised CfTXN2H6-Cys44Ser. Modelling suggests Arg45 (44), Glu73 (72), the Ile110 (109) cis-Pro111 (110)-bond and Arg129 (128) to be involved in the binding of trypanothione to CfTXN2 (CfTXN1). The model of TXN-substrate interaction is consistent with functional characteristics of known and newly designed muteins (CfTXN2H6-Arg129Asp and Glu73Arg) and the 1N-glutathionyl-spermidine binding in the CfTXN2H6-Cys44Ser structure.

Convenient isolation and kinetic mechanism of glutathionylspermidine synthetase from Crithidia fasciculata.

Trypanothione, the essential metabolite in the oxidant defense system of trypanosomatids, is synthesized by two distinct proteins, glutathionylspermidine synthetase and trypanothione synthetase. Glutathionylspermidine synthetase was purified to homogeneity from the trypanosomatid Crithidia fasciculata by aqueous two-phase systems and chromatography. The enzyme showed a specific activity of 38 micromol of glutathionylspermidine formed per min per mg of protein. Its molecular mass was 78 kDa in SDS-polyacrylamide gel electrophoresis, and it appeared predominantly monomeric in native polyacrylamide gel electrophoresis and gel filtration. The isoelectric point was at pH 4.6, and the pH optimum was near 7.6. Partial amino acid sequencing revealed homology with, but low similarity to, the glutathionylspermidine synthetase/amidase of Escherichia coli, and amidase activity was not detected in glutathionylspermidine synthetase of C. fasciculata. The kinetics of trypanosomatid glutathionylspermidine synthetase revealed a rapid equilibrium random mechanism with limiting Km values for Mg2+-ATP, GSH, and spermidine of 0.25 +/- 0.02, 2.51 +/- 0.33, and 0.47 +/- 0. 09 mM, respectively, and a kcat of 415 +/- 78 min-1. Partial reactions at restricted cosubstrate supply were not detected by 31P NMR, supporting the necessity of a quarternary complex formation for catalysis. ADP inhibited competitively with respect to ATP (Ki = 0. 08 mM) and trypanothione exerted a feedback inhibition competitive with GSH (Ki = 0.48 mM).

Lipase of Pseudomonas cepacia for biotechnological purposes: purification, crystallization and characterization.

Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose. The eluted lipase crystallized spontaneously at 4 degrees C in the eluent, containing 58-69% 2-propanol. The yield of the lipase was 87-100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein. This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its purity was determined by SDS-PAGE and capillary zone electrophoresis to be > or = 99%. Immobilization on Sepharose increased its stability in organic solvents. This lipase of P. cepacia differs from that of other Pseudomonas strains in respect to substrate specificity and during crystallization. It exhibits a high stability in organic solvents and supercritical carbon dioxide.

Screening, purification and properties of a thermophilic lipase from Bacillus thermocatenulatus.

By screening of 15 thermophilic Bacillus strains, five strains exhibiting lipase activity were found. Among these the strain Bacillus thermocatenulatus (DSM 730) produced the highest lipase activity. The lipase proved to be inducible and extracellular and was purified 67-fold to homogenous state by hexane extraction, methanol precipitation and ion-exchange chromatography on Q-Sepharose. The molecular weight of the lipase determined by SDS-PAGE is 16 kDa. However, the lipase forms very large aggregates (> 750 kDa) as observed after native PAGE, which makes handling of the lipase very difficult. The lipase binds almost irreversibly on different chromatography matrices, e.g., Amberlite and Serolite, and is very stable in the immobilised form. The N-terminal sequence consists of 53% apolar amino acids and shows no significant homology towards other known lipase sequences. Maximum activity was found at pH 7.5-8.0 and 60-70 degrees C with pNPP and olive oil as substrates.

The 92-kDa chitinase from Streptomyces olivaceoviridis contains a lysine-C endoproteinase at its N-terminus.

Serine proteinases of 42, 22 and 14 kDa were purified from the culture fluid of Streptomyces olivaceoviridis by FPLC. The first 14 amino acids at their N-termini were identical and coincide with the N-terminal amino acid sequence of 92-kDa chitinase, which was found to hydrolyse casein. The four proteins hydrolyse synthetic substrates at the carboxyl group of lysine and (more slowly) arginine. The 14-kDa endoproteinase releases only two fragments of 42 and 43 kDa from beta-galactosidase. When the pure 92-kDa chitinase was incubated at 37 degrees C in Tris.HCl buffer, it was cleaved into a 70-kDa chitinase and a 22-kDa proteinase which in its part is rapidly degraded to a 14-kDa proteinase.

Kinetic study of lipase catalyzed esterification reactions in water-in-oil microemulsions.

The kinetics of the esterification of lauric acid by (-)menthol, catalyzed by Penicillium simplicissimum lipase, was studied in water/bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT)/isooctane microemulsions. Due to their low water content, microemulsions assist in reversing the direction of lipase activity, favoring synthetic reactions. The kinetics of this synthesis follows a Ping-Pong Bi--Bi mechanism. The values of all apparent kinetic parameters were determined. The theoretical model for the expression of enzymic activity in reverse micelles, proposed by Verhaert et al. (Verhaert, R., Hilhorst, R., Vermüe, M., Schaafsma, T. J., Veeger, C. 1990. Eur. J. Biochem. 187: 59-72) was extended to express the lipase activity in an esterification reaction involving two hydrophobic substrates in microemulsion systems. The model takes into account the partitioning of the substrates between the various phases and allows the calculation of the intrinsic kinetic constants. The experimental results showing the dependence of the initial velocity on the hydration ratio, W(o) = [H(2)O]/[AOT], of the reverse micelles, were in accordance with the theoretically predicted pattern.

Purification and properties of a lipase from Penicillium expansum.

Penicillum expansum DSM 1994 produces a new, inducible extracellular lipase when grown in medium containing 0.1% olive oil. Maximum activity was obtained after 4 days of incubation at 20 degrees C. The enzyme was purified 219-fold by cross-flow filtration, ammonium sulfate precipitation and hydrophobic interaction chromatography to a final specific activity of 558 U/mg. The molecular weight of the homogeneous lipase was (25 kDa) determined by gel filtration and SDS-PAGE, however, it forms active dimers and higher aggregates as observed after native PAGE. The enzyme was identified as a glycoprotein with a pI of 5.5. The N-terminal sequence shows a homology to sequences of other lipase just behind their consensus sequence. Enzyme stability was enhanced by the addition of Tween 20 and Lubrol PX. The enzyme showed a maximum activity at pH 9 at 45 degrees C and was stable at a broad pH range of 6-10. Lipase of P. expansum showed a preference for triacylglycerols, but no positional specificity.

Chitinases of Streptomyces olivaceoviridis and significance of processing for multiplicity.

Five extracellular chitinases of 20.5, 30, 47, 70, and 92 kDa purified from the culture filtrate of Streptomyces olivaceoviridis ATCC 11238 differed in their sequences at the amino termini of the protein chains. In the native state, the chitinases were found to be resistant to proteolysis by trypsin, papain, and Staphylococcus aureus V8 protease. The latter produced several fragments of identical molecular mass from chitinases denaturated with sodium dodecyl sulfate. Five proteases were detected in the protein concentrate from the culture filtrate, and two of them showing ability to cleave chitinases in the native state were purified. One, a protease of 42 kDa, released a 30-kDa protein from the 70-kDa chitinase that reacts with anti-30 kDa chitinase antibodies; the other, a protease of 29 kDa, split the 30-kDa chitinase into 20.5-, 18-, and 16-kDa fragments. From these results, it was deduced that the 70-kDa chitinase is the precursor protein of the 30- and 20.5-kDa chitinases.

Purification and properties of lipase from Penicillium simplicissimum.

A Penicillium simplicissimum strain has been found to produce an inducible extracellular lipase. Triolein was the best inducer for the enzyme production with the highest activity being achieved after 48 h of incubation. The purified lipase showed a molecular weight of 56,000 by SDS-PAGE. The enzyme exhibited a high ratio of apolar amino acids. The lipase was stable in the pH range of 5-7 and at 50 degrees C for 15 min. The optimum assay conditions were 37 degrees C and pH 5.0. The enzyme showed a high stability in water immiscible organic solvents. Lipase from P. simplicissimum is nonspecific and hydrolyses each of the three bonds of triacylglycerols.

Purification of proteins from cell culture supernatants.

Desired proteins excreted by animal cells usually reach rather low concentrations in the culture supernatant and have to be purified from an excess of serum proteins which are added to the animal cell culture to maintain its viability and/or productivity. Defined media offer among others the advantage of an easier purification procedure. In addition lysis of cells may contribute also to the complexity of the protein mixture encountered. If fetal calf serum or new born calf serum is used in cultivation, bovine serum albumin and globulins will be the most abundant protein in the supernatant. The interaction of albumin especially with hydrophobic proteins represents significant problems for the effective purification of minor constituent. Isolation of a desired protein follows the general scheme: concentration: by precipitation, ultrafiltration, batch adsorption or partition in aqueous phase system; enrichment: by chromatography or partition; high resolution purification: by chromatography and/or immuno adsorption; final concentration and finishing: pyrogen removal, sterilization, formulation. Biochemical engineering aspects of proteins purification are discussed using human interferon-beta produced in a recombinant mouse cell line as an example. The process developed encloses extraction of interferon-beta from the production medium in aqueous two-phase systems and subsequent recovery of interferon-beta from the top phase by high pressure liquid chromatography.

[Significance of pCO2 for cerebral blood flow in neonatology. A Doppler sonographic study]. / Die Bedeutung des pCO2 für die Hirndurchblutung in der Neonatologie. Eine Doppler-sonografische Untersuchung.

Range-gated pulsed Doppler-ultrasonographic blood flow measurements of 3 cerebral arteries (a. cerebri anterior, a. carotis interna, a. basilaris) were performed during acute pCO2-changes in 16 neonates treated in an intensive care unit. Pulsatility index, peak systolic and end diastolic flow velocity, and mean velocity were evaluated. Mean velocity in the a. carotis interna and the a. basilaris depends on the pCO2, and increases resp. decreases by 5.6% per mm Hg of rise resp. fall of the pCO2. Assuming no pCO2-dependent diameter changes of the large cerebral arteries volume flow would increase resp. decrease by a proportional amount. When obtaining qualitative or semiquantitative informations about volume flow by Doppler-ultrasonography anatomic conditions must be considered carefully.

Purification of human fibroblast interferon by extraction in aqueous two-phase systems.

Liquid-liquid extraction offers a new method for the concentration and purification of human fibroblast interferon (IFN-beta). Different derivatives of polyethylene glycol (PEG)--liquid ion exchangers or affinity ligands--can be effectively used for the extraction of IFN-beta in combination with Dextran-T 500 or orthophosphate. Important parameters for the partition of IFN-beta have been investigated, e.g., the influence of the concentrations of the phase-forming components, the addition of salts, the volume ratio of the top and bottom phases, and the content of crude IFN-beta. Systems containing polyethylene glycol-phosphate ester/orthophosphate/sodium chloride (1/19.5/2.9% w/w at pH 6.9 or 2/19/7.5% w/w at pH 5-5.9, respectively) resulted in up to 350-fold purified IFN-beta in the top phases with a yield of 74-100% and a specific activity of 3-7 X 10(6) units/mg. The efficiency of extraction in the aqueous phase system is a consequence of an extremely limited solubility of IFN-beta in the bottom phase. One of the advantages of the new method is that it is independent of the process volume and can be performed easily and quickly.

Monoclonal antibodies against human fibroblast interferon.

A stable hybridoma clone derived by infusion of mouse myeloma cells (cell line Fo) and spleen cells of immunized mice has been isolated which secretes monoclonal antibodies against human fibroblast interferon (interferon-beta). The antibody inhibits the antiviral activity of human fibroblast interferon in an antiviral assay using human FS4 fibroblast, reacts immunologically with interferon-beta separated by sodium dodecylsulfate/polyacrylamide gel electrophoresis and subsequent transfer to nitrocellulose and absorbs interferon-beta immunologically when bound to CNBr-activated Sepharose. It also inhibits the antiviral activity of human fibroblast interferon-beta from which the sugar moiety has been cleaved off by enzymatic treatment. The antibody is therefore probably directed against the protein moiety of the interferon molecules.

[Formamidase--microheterogeneity, catalytic properties and inhibitors (author's transl)]. / Formamidase- Untersuchungen zu Mikroheterogenität, katalytischen Eigenschaften und Inhibitoren.

Formamidase from rat liver proved to be microheterogenous. After preparative isoelectric focusing in density gradient columns, two peaks of formamidase with identical substrate specificity were identified. By analytical focusing in thin layers of polyacrylamide or Sephadex G-75 SF, even five bands could be separated. Their isoelectric points were 4.75, 4.78, 4.82, 4.92 (main band) and 5.11, but their Michaelis constants did not differ significantly (54 to 62 mumol/l). An identical molecular weight of 34700 +/- 3200 for all bands was determined by disc electrophoresis. This value was confirmed by sedimentation analyses (so20,w = 3.00 S) and electrophoresis in the presence of sodium dodecyl-sulfate (Mr 34900 +/- 2300), which only gave a single band. The homogeneity was also confirmed by electrophoresis in the presence of 6M urea. Repeated disc electrophoresis of focusing under native conditions with single, isolated formamidases again resulted in different bands which were identified, not only by Coomassie Blue, but also by their hydrolytic cleavage of naphthyl acetate. Formamidase showed neither proteolytic nor asparagine-amidohydrolase activity and oligosaccharide conjugates were not detectable. Ampholytes, buffer ions, pH and peroxodisulfate did not affect the heterogeneity. "Initial burst" measurements with diethyl(4-nitrophenyl) phosphate yielded an equivalent weight of 36,300. Formylkynurenine reduced this inhibition very effectively. Thus, an extraordinary reactive serine residue appeared to be located in the catalytic site of formamidase. A participation of sulfhydrylgroups in the inactivating reaction of arsenite was excluded although two such groups were detected by 5,5'-dithiobis(2-nitrobenzoic acid). N-Bromosuccinimide reacted primarily with one of the nine tryptophan residues without loss of enzymatic activity, but a 18.6-fold excess of this reagent resulted in a complete loss of activity. The reaction rates of the most effective inhibitors and of the protective action of formylkynurenine were determined. Thus, formamidase must clearly be distinguished from typical serine esterases and proteases.

Purification and some properties of formamidase from rat and pig liver.

Formamidase was purified 4700-fold from rat liver cytoplasma. Its main molecular and catalytic properties are reported. In pig liver three active forms were observed.