BackgroundCultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in Escherichia coli in simple shaken cultures.ResultsThe enzymatic glucose release system together with a well-balanced combination of mineral salts and complex medium additives provided high cell densities, high protein yields and a considerably improved proportion of soluble proteins in harvested cells. The cultivation method consists of three steps: 1) controlled growth by glucose-limited fed-batch to OD600 ~10, 2) addition of growth boosters together with an inducer providing efficient protein synthesis within a 3 to 6 hours period, and 3) a slow growth period (16 to 21 hours) during which the recombinant protein is slowly synthesized and folded. Cell densities corresponding to 10 to 15 g l-1 cell dry weight could be achieved with the developed technique. In comparison to standard cultures in LB, Terrific Broth and mineral salt medium, we typically achieved over 10-fold higher volumetric yields of soluble recombinant proteins.ConclusionsWe have demonstrated that by applying the novel EnBase® Flo cultivation system in shaken cultures high cell densities can be obtained without impairing the productivity per cell. Especially the yield of soluble (correctly folded) proteins was significantly improved in comparison to commonly used LB, Terrific Broth or mineral salt media. This improvement is thought to result from a well controlled physiological state during the whole process. The higher volumetric yields enable the use of lower culture volumes and can thus significantly reduce the amount of time and effort needed for downstream processing or process optimization. We claim that the new cultivation system is widely applicable and, as it is very simple to apply, could widely replace standard shake flask approaches.

Novel phenylazole ligands were applied successfully in the synthesis of cyclometalated iridium(III) complexes of the general formula [Ir(phenylazole)(2)(bpy)]PF(6) (bpy=2,2'-bipyridine). All complexes were fully characterized by NMR, IR, and MS spectroscopic studies as well as by cyclic voltammetry. Three crystal structures obtained by X-ray analysis complemented the spectroscopic investigations. The excited-state lifetimes of the iridium complexes were determined and showed to be in the range of several hundred ns to multiple µs. All obtained iridium complexes were active as photosensitizers in catalytic hydrogen evolution from water in the presence of triethylamine as a sacrificial reducing agent. Applying an in situ formed iron-based water reduction catalyst derived from [HNEt(3)](+) [HFe(3)(CO)(11)](-) and tris[3,5-tris-(trifluoromethyl)-phenyl]phosphine as the ligand, [Ir(2-phenylbenz-oxazole)(2)-(bpy)]PF(6) proved to be the most efficient complex giving a quantum yield of 16% at 440 nm light irradiation.

Prolyl endopeptidase (PEP) is a proline-specific oligopeptidase with a reported effect on learning and memory in different rat model systems. Using the astroglioma cell line U343, PEP expression was reduced by an antisense technique. Measuring different second-messenger concentrations revealed an inverse correlation between inositol 1,4,5-triphosphate [Ins(1,4,5)P 3 ] concentration and PEP expression in the generated antisense cell lines. However, no effect on cAMP generation was observed. In addition, complete suppression of PEP activity by the specific inhibitor, Fmoc-Ala-Pyrr-CN (5 lM) induced in U343 and other cell lines an enhanced, but delayed, increase in Ins(1,4,5)P 3 concentration. This indicates that the proteolytic activity of PEP is responsible for the observed effect. Furthermore, the reduced PEP activity was found to amplify Substance P-mediated stimulation of Ins(1,4,5)P 3 . The effect of reduced PEP activity on second-messenger concentration indicates a novel intracellular function of this peptidase, which may have an impact on the reported cognitive enhancements due to PEP inhibition.Keywords: antisense; inositol 1,4,5-triphosphate; prolyl endopeptidase; protease; second messenger; Substance P.Prolyl endopeptidase (PEP; also called prolyl oligopeptidase) is a serine peptidase characterized by oligopeptidase activity. It is grouped in family S9A in clan SC [1]. Enzymes belonging to clan SC are distinct from trypsin-type and subtilisin-type serine peptidases in their structure and the order of the catalytic triad residues in the primary sequence [2,3]. The recently reported three-dimensional structure of PEP revealed a two-domain organization [4]. The catalytic domain displays an a/b hydrolase fold in which the catalytic triad (Ser554, His680, Asp641) is covered by a so-called b-propeller domain. The propeller domain probably controls the access of potential substrates to the active site of the enzyme and excludes peptides containing more than 30 amino acids.Although the enzymatic and structural properties of PEP are well known, its biological function is far from being fully understood [5,6]. Highly conserved in mammals, it is ubiquitously distributed, with high concentrations in the brain [7]. Recently, the enzyme became of pharmaceutical interest because of a reported cognitive enhancement induced by treatment with specific PEP inhibitors [8,9]. In rats displaying scopolamine-induced amnesia, PEP inhibition caused acetylcholine release in the frontal cortex and hippocampus [10]. Furthermore, administration of a PEP inhibitor to rats with middle cerebral artery occlusion prolonged passive avoidance latency and reduced the prolonged escape latency in the Morris water maze task [11]. The potential of PEP inhibitors as antidementia drugs was further supported by reports of neuroprotective effects. Inducing neurodegeneration in cerebellar granule cells led to increased neuronal survival and enhanced neurite outgrowth in the presence of a PEP inhibitor [12]. Moreover, the level of m 3 -muscarinic ace...

The ultrafast injection dynamics, early recombination dynamics, and spectral signatures of four systematically varied dye-metal oxide hybrid systems were investigated using transient absorption spectroscopy techniques. First, photoinduced electron transfer from two different perylene derivatives into zinc oxide (ZnO) colloidal films is reported. Here, the electronic coupling of the perylene chromophore 2,5-Di-tert-butyl-perylene-9-yl-propionic acid (1) to the ZnO colloids was weaker than the electronic coupling of the chromophore 2,5-Di-tert-butyl-perylene-9-yl-acrylic acid (2). Second, the photoinduced electron transfer of the same two molecules attached to TiO2 colloids was measured and compared to the results for the ZnO colloids using the same techniques. The temporal traces at both the excited-state and the cationic state of the chromophores attached to the semiconductor surfaces were measured simultaneously and showed very good agreement, which indicated a direct injection into the semiconductor. The overall injection times for the ZnO samples was as short as 190 fs, which suggested a strong electronic coupling element for these systems. This injection time is short compared to reports on similar ZnO hybrid systems, but it is still longer than the injection times reported for the TiO2 hybrid systems. The transient absorption spectra of molecule 2 attached to TiO2 showed a large negative signal at 530–550 nm, which indicated the presence of a direct charge transfer state contribution in this system.

Controlled initiation of biochemical events and in particular of protein activity is a powerful tool in biochemical research. Specifically, optical trigger signals are an attractive approach for remote control of enzyme activity. We present a method for generating optical control of enzyme activity applicable to a widespread range of enzymes. The approach is based on short laser pulses as optical "switches" introducing an instantaneous change of the pH-value for activation of protein function. The pH-jump is induced by proton release from 2-nitrobenzaldehyde. Reaction conditions were chosen to yield a pH-jump of almost 3 units on switching from inactive to active conditions for the enzyme. In this experimental setup, irradiation can be realized without any loss of enzyme activity. Following this change in pH-value, a controlled activation of hydrolytic activity of acid phosphatase is successfully demonstrated. This application provides a general method for photocontrol of enzymatic function for proteins having a significant pH-profile. The kinetic data for the substrate 6-chloro-8-fluoro-4-methylumbelliferone phosphate are determined.

The reaction pathways of bis-(2-phenylpyridinato-)(2,2'-bipyridine)iridium(III)hexafluorophosphate [Ir(ppy)2(bpy)]PF6 within a photocatalytic water reduction system for hydrogen generation based on an iron-catalyst were investigated by employing time-resolved photoluminescence spectroscopy and time-dependent density functional theory. Electron transfer (ET) from the sacrificial reagent to the photoexcited Ir complex has a surprisingly low probability of 0.4% per collision. Hence, this step limits the efficiency of the overall system. The calculations show that ET takes place only for specific encounter geometries. At the same time, the presence of the iron-catalyst represents an energy loss channel due to a triplet-triplet energy transfer of Dexter type. This loss channel is kept small by the employed concentration ratios, thus favoring the reductive ET necessary for the water reduction. The elucidated reaction mechanisms underline the further need to improve the sun light's energy pathway to the catalyst to increase the efficiency of the photocatalytic system.

The photoreduction of the bis(2-phenylpyridinato-)(2,2'-bipyridine)iridium(III) ion ([Ir(ppy)2(bpy)](+)), used as a photosensitizer in photocatalytic water splitting, by triethylamine was studied by means of UV/VIS, XANES, and EPR spectroscopies, supported by theoretical calculations at density functional theory (DFT) and complete active space self-consistent field (CASSCF/CASPT2) levels. The combination of these methods suggests a predominant bpy localization of the spin-density of the unpaired electron with notable delocalization to the Ir center. This is particularly evident from EPR and theoretical results and leads to broad EPR lines and a large anisotropy of the g-factor.

While the nutrient limited fed-batch technology is the standard of the cultivation of microorganisms and production of heterologous proteins in industry, despite its advantages in view of metabolic control and high cell density growth, shaken batch cultures are still the standard for protein production and expression screening in molecular biology and biochemistry laboratories. This is due to the difficulty and expenses to apply a controlled continuous glucose feed to shaken cultures. New ready-made growth media, e.g. by biocatalytic release of glucose from a polymer, offer a simple solution for the application of the fed-batch principle in shaken plate and flask cultures. Their wider use has shown that the controlled diet not only provides a solution to obtain significantly higher cell yields, but also in many cases folding of the target protein is improved by the applied lower growth rates; i.e. final volumetric yields for the active protein can be a multiple of what is obtained in complex medium cultures. The combination of the conventional optimization approaches with new and easy applicable growth systems has revolutionized recombinant protein production in Escherichia coli in view of product yield, culture robustness as well as significantly increased cell densities. This technical development establishes the basis for successful miniaturization and parallelization which is now an important tool for synthetic biology and protein engineering approaches. This review provides an overview of the recent developments, results and applications of advanced growth systems which use a controlled glucose release as substrate supply.