Tailoring the properties of (catalytically)-active inclusion bodies.

BACKGROUND: Immobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobilization usually includes additional cross-linking or adsorption steps. We have previously shown in a few case studies that the molecular biological fusion of an aggregation-inducing tag to a target protein induces the intracellular formation of protein aggregates, so called inclusion bodies (IBs), which to a certain degree retain their (catalytic) function. This enables the combination of protein production and immobilization in one step. Hence, those biologically-produced immobilizates were named catalytically-active inclusion bodies (CatIBs) or, in case of proteins without catalytic activity, functional IBs (FIBs). While this strategy has been proven successful, the efficiency, the potential for optimization and important CatIB/FIB properties like yield, activity and morphology have not been investigated systematically. RESULTS: We here evaluated a CatIB/FIB toolbox of different enzymes and proteins. Different optimization strategies, like linker deletion, C- versus N-terminal fusion and the fusion of alternative aggregation-inducing tags were evaluated. The obtained CatIBs/FIBs varied with respect to formation efficiency, yield, composition and residual activity, which could be correlated to differences in their morphology; as revealed by (electron) microscopy. Last but not least, we demonstrate that the CatIB/FIB formation efficiency appears to be correlated to the solvent-accessible hydrophobic surface area of the target protein, providing a structure-based rationale for our strategy and opening up the possibility to predict its efficiency for any given target protein. CONCLUSION: We here provide evidence for the general applicability, predictability and flexibility of the CatIB/FIB immobilization strategy, highlighting the application potential of CatIB-based enzyme immobilizates for synthetic chemistry, biocatalysis and industry.

Initial characterization of a blue-light sensing, phototropin-related protein from Pseudomonas putida: a paradigm for an extended LOV construct.

The open reading frame PP2739 from Pseudomonas putida KT2440 encodes a 151 amino acid protein with sequence similarity to the LOV domains of the blue-light sensitive protein YtvA from Bacillus subtilis and to the phototropins (phot) from plants. This sensory box LOV protein, PpSB2-LOV, comprises a LOV core, followed by a C-terminal segment predicted to form an alpha-helix, thus constituting a naturally occurring paradigm for an extended LOV construct. The recombinant PpSB2-LOV shows a photochemistry very similar to that of YtvA and phot-LOV domains, yet the lifetime for the recovery dark reaction, taurec=114 s at 20 degrees C, resembles that of phot-LOV domains (5-300 s) and is much faster than that of YtvA or YtvA-LOV (>3000 s). Time-resolved optoacoustics reveals phot-like, light-driven reactions on the ns-micros time window with the sub-nanosecond formation of a flavin triplet state (PhiT=0.46) that decays into the flavin-cysteine photoadduct with 2 micros lifetime (Phi390=0.42). The fluorescence spectrum and lifetime of the conserved W97 resembles the corresponding W103 in full-length YtvA, although the quantum yield, PhiF, is smaller (about 55% of YtvA) due to an enhanced static quenching efficiency. The anisotropy of W97 is the same as for W103 in YtvA (0.1), and considerably larger than the value of 0.06, found for W103 in YtvA-LOV. Different to YtvA and YtvA-LOV, the fluorescence for W97 becomes larger upon photoproduct formation. These data indicate that W97 is located in a similar environment as W103 in full-length YtvA, but undergoes larger light-driven changes. It is concluded that the protein segment located C-terminally to the LOV core (analogous to an interdomain linker) is enough to confer to the conserved tryptophan the fluorescence characteristics typical of full-length YtvA. The larger changes experienced by W97 upon light activation may reflect a larger conformational freedom of this protein segment in the absence of a second domain.

[Severe parachuting accident. Analysis of 122 cases]. / Der schwerwiegende Gleitschirmunfall. Analyse von 122 Fällen.

Based on a population of 122 severely injured patients the causes of paragliding accidents and the patterns of injury are analyzed. A questionnaire is used to establish a sport-specific profile for the paragliding pilot. The lower limbs (55.7%) and the lower parts of the spine (45.9%) are the most frequently injured parts of the body. There is a high risk of multiple injuries after a single accident because of the tremendous axial power. The standard of equipment is good in over 90% of the cases. Insufficient training and failure to take account of geographical and meteorological conditions are the main determinants of accidents sustained by paragliders, most of whom are young. Nevertheless, 80% of our patients want to continue paragliding. Finally some advice is given on how to prevent paragliding accidents and injuries.

[Diagnosis of kidney transplant rejection. The value of scintigraphy, sonography and NMR tomography]. / Diagnostik von Nierentransplantatabstossungen. Stellenwert von Szintigraphie, Sonographie und Kernspintomographie.

Scintigraphy, ultrasound and magnetic resonance imaging were undertaken within three days on 73 patients who had had renal transplantation. Clinical and histological criteria were used to assess the ability of diagnosing rejection of the transplant. In all instances of rejection cortex-marrow differentiation was absent on magnetic resonance (MR) imaging; there were no false-negative results. Sensitivity as to rejection was 100% for MR imaging, 58% for scintigraphy and 32% for ultrasound. It may be possible to distinguish between cytomegalovirus infection and rejection reaction by MR imaging. It is concluded that MR imaging is the most reliable method for predicting rejection reactions, while ultrasound and scintigraphy are valuable for making other diagnostic decisions.

Venom-specific IgE and IgG antibodies as a measure of the degree of protection in insect-sting-sensitive patients.

Venom-specific IgE and IgG antibodies were measured in the sera of bee-venom-sensitive patients during a 3-year hyposensitization period. The level of specific IgG antibodies initially increased, and 2 months after the start of therapy, this increase was on average five-fold. A concomitant but non-significant increase in specific IgE antibodies was also observed initially. Later during the treatment period specific IgE antibodies showed a continuous decline and after 3 years the level was one third of the pre-treatment level. Venom-specific IgG antibodies, however, remained above the pre-treatment level. The disappearance of the severe allergic reactions was related to the level of both IgE and IgG antibodies as demonstrated in seventy-six bee sting challenges. In patients with specific IgG antibody levels above 400 u/ml, no severe reactions were observed even if the patient had levels of specific IgE antibodies of RAST class 3 or 4. These data suggest that the relationship between venom-specific IgE and IgG antibodies permits an evaluation of the state of immunity to insect stings.