Improved methods of cultivation and production of deuteriated proteins from E. coli strains grown on fully deuteriated minimal medium

AIMS: The aim was to develop reliable and economical protocols for the production of fully deuteriated biomolecules by bacteria. This required the preparation of deuterium-tolerant bacterial strains and an understanding of the physiological mechanisms of acquisition of deuterium tolerance. METHODS AND RESULTS: We report here improved methods for the cultivation of Escherichia coli on fully deuteriated minimal medium. A multi-stage adaptation protocol was developed; this included repeated plating and selection of colonies and resulted in highly deuterium-tolerant cell cultures. Three E. coli strains, JM109, MRE600 and MRE600Rif, were adapted to growth on deuteriated succinate medium. This is the first report of JM109 being adapted to deuteriated minimal media. The adapted strains showed good, consistent growth rates and were capable of being transformed with plasmids. Expression of heterologous proteins in these strains was reliable and yields were consistently high (100-200 mg l-1). We also show that all E. coli cells are inherently capable of growth on deuteriated media. CONCLUSIONS: We have developed a new adaptation protocol that resulted in three highly deuterium-tolerant E. coli strains. Deuterium-adapted cultures produced good yields of a deuteriated recombinant protein. We suggest that E. coli cells are inherently capable of growth on deuteriated media, but that non-specific mutations enhance deuterium tolerance. Thus plating and selection of colonies leads to highly deuterium-tolerant strains. SIGNIFICANCE AND IMPACT OF STUDY: An understanding of the mechanism of adaptation of E. coli to growth on deuteriated media allows strategies for the development of disabled deuterium-tolerant strains suitable for high-level production of deuteriated recombinant proteins and other biomolecules. This is of particular importance for nuclear magnetic resonance and neutron scattering studies of biomolecules.     

Predictive markers of safety and immunogenicity of adjuvanted vaccines

Vaccination represents one of the greatest public health triumphs; in part due to the effect of adjuvants that have been included in vaccine preparations to boost the immune responses through different mechanisms. Although a variety of novel adjuvants have been under development, only a limited number have been approved by regulatory authorities for human vaccines. This report reflects the conclusions of a group of scientists from academia, regulatory agencies and industry who attended a conference on the current state of the art in the adjuvant field. Held at the U.S. Pharmacopeial Convention (USP) in Rockville, Maryland, USA, from 18 to 19 April 2013 and organized by the International Association for Biologicals (IABS), the conference focused particularly on the future development of effective adjuvants and adjuvanted vaccines and on overcoming major hurdles, such as safety and immunogenicity assessment, as well as regulatory scrutiny. More information on the conference output can be found on the IABS website, http://www.iabs.org/.     

Burkholderia pseudomallei γ-carbonic anhydrase is strongly activated by amino acids and amines

Activation of the γ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei (BpsγCA) with a series of natural and non-natural amino acids and aromatic/heterocyclic amines has been investigated. The best BpsγCA activators were d-His, l-DOPA, d-Trp, 4-amino-l-Phe, dopamine, 2-(2-aminoethyl)pyridine, 2-aminoethyl-piparazine/morpholine and l-adrenaline, which showed activation constants ranging between 9 and 86 nM. The least effective activators were l-His, l-Phe and 2-pyridyl-methylamine, with K_As in the range of 1.73–24.7 μM. As little is known about the role of γ-CAs in the lifecycle and virulence of this saprophytic bacterium, this study may shed some light on such phenomena. This is the first CA activation study of a γ-CA from a pathogenic bacterium, the only other such study being on the enzyme discovered in the archaeon Methanosarcina thermophila, Cam.     

Critical residues for RNA discrimination of the histone hairpin binding protein (HBP) investigated by the yeast three-hybrid system

The histone hairpin binding protein (HBP, also called SLBP, which stands for stem-loop binding protein) binds specifically to a highly conserved hairpin structure located in the 3' UTR of the cell-cycle-dependent histone mRNAs. HBP consists of a minimal central RNA binding domain (RBD) flanked by an N- and C-terminal domain. The yeast three-hybrid system has been used to investigate the critical residues of the human HBP involved in the binding of its target hairpin structure. By means of negative selections followed by positive selections, we isolated mutant HBP species. Our results indicate tight relationships between the RBD and the N- and C-terminal domains.     

A viscometric study of the biodegradation of photographic gelatin by fungi isolated from cinematographic films

The biodegradation of photographic gelatin grade (Bloom 225) material was studied by viscometry in aqueous solution (at 37 °C, 6.67% w/w) using filamentous fungi isolated and identified from cinematographic film stored in different Spanish archives. From viscosity data, different variables such as molecular weight and chain scission were calculated. To ensure initial spore suspension concentration was standardized for all the biodegradation experiments, a correlation between transmittance at 530 nm of fungal spore suspensions and the corresponding cytometric determination of populations was established for all the fungal strains studied in this work. The bioassay experiments were carried out at 25 and 4 °C using an initial concentration of fungi of 4.5×10⁵ conidia/mL except in the case of the genus Alternaria, where the concentration was 10 times lower. The fungal strains were three species of Aspergillus, i.e., A .ustus, A. nidulans var. nidulans, A. versicolor, seven Penicillium chrysogenum strains, and Cladosporium cladosporioides, Alternaria alternata, Mucor racemosus, Phoma glomerata, and Trichoderma longibrachiatum. All were gelatinase positive. Through the viscosity decay profiles with bioassay-time and the corresponding calculated chain scission, the relative quantitative gelatinase efficiency of these fungi has been evaluated.     

The Mr 17500 region of the A chain of urokinase is required for interaction with a specific receptor in A431 cells

We have found the existence of specific receptors for the plasminogen activator, urokinase, in A431 human epidermoid carcinoma cells, cultures in plasminogen-free conditions. Two subsets of receptors have been recognized on the basis of 125I-labelled urokinase binding analysis: about 1 X 10(3) high-affinity (Kd = 5.0 X 10(-11) M) and 1 X 10(5) low-affinity (Kd = 9 X 10(-9) M) receptors per cell. The electron microscopic observation of a urokinase: ferritin conjugate has shown single and clustered receptors at the cell surface. Down-regulation of the receptors (T1/2 = 3.77 h) follows the binding of urokinase. The binding does not involve an intact catalytic site and is inhibited by a monoclonal antibody against the Mr 17500 proteolytic fragment of the A chain of urokinase.