Efficient phage display of intracellularly folded proteins mediated by the TAT pathway

Phage display with filamentous phages is widely applied and well developed, yet proteins requiring a cytoplasmic environment for correct folding still defy attempts at functional display. To extend applicability of phage display, we employed the twin-arginine translocation (TAT) pathway to incorporate proteins fused to the C-terminal domain of the geneIII protein into phage particles. We investigated functionality and display level of fluorescent proteins depending on the translocation pathway, which was the TAT, general secretory (SEC) or signal recognition particle (SRP) pathway mediated by the TorA, PelB or DsbA signal sequences, respectively. Importantly, for green fluorescent protein, yellow fluorescent protein and cyan fluorescent protein, only TAT, but not SEC or SRP, translocation led to fluorescence of purified phage particles, although all three proteins could be displayed regardless of the translocation pathway. In contrast, the monomeric red fluorescent protein mCherry was functionally displayed regardless of the translocation pathway. Hence, correct folding and fluorophor formation of mCherry is not limited to the cytosol. Furthermore, we successfully displayed firefly luciferase as well as an 83 kDa argonaute protein, both containing free cysteines. This demonstrates broad applicability of the TAT-mediated phagemid system for the display of proteins requiring cytoplasmic factors for correct folding and should prove useful for the display of proteins requiring incorporation of co-factors or oligomerization to gain function.     

EGFR-Binding Peptides: From Computational Design towards Tumor-Targeting of Adeno-Associated Virus Capsids

The epidermal growth factor receptor (EGFR) plays a central role in the progression of many solid tumors. We used this validated target to analyze the de novo design of EGFR-binding peptides and their application for the delivery of complex payloads via rational design of a viral vector. Peptides were computationally designed to interact with the EGFR dimerization interface. Two new peptides and a reference (EDA peptide) were chemically synthesized, and their binding ability characterized. Presentation of these peptides in each of the 60 capsid proteins of recombinant adeno-associated viruses (rAAV) via a genetic based loop insertion enabled targeting of EGFR overexpressing tumor cell lines. Furthermore, tissue distribution and tumor xenograft specificity were analyzed with systemic injection in chicken egg chorioallantoic membrane (CAM) assays. Complex correlations between the targeting of the synthetic peptides and the viral vectors to cells and in ovo were observed. Overall, these data demonstrate the potential of computational design in combination with rational capsid modification for viral vector targeting opening new avenues for viral vector delivery and specifically suicide gene therapy.     

Matrix models of discretely bending, stiff polymers

Polymer models which make use of the Ising model and transfer matrix techniques remind us, for example, of the work of Flory [Statistical mechanics of chain molecules, 1969] and Zimm and Bragg [J Chem Phys, 31 (1959) 526]. We investigate the properties of some such polymer models where the chain conformation can be described solely by an Ising-like parameterization and a set of independent, predetermined bond direction vectors or by a Potts-like model for directions of bond vectors on a lattice, with the specific aim of understanding more closely the connection of constraints and forces on the chain ends for polymers which, in general, are of arc length corresponding to their persistence lengths. Instances of these models are directed helical walks, random sequential walks, bimodally distributed in direction walks or relatively short, stiff chains fixed into a network. The behavior of this model under deformation in statistical mechanics and its dynamical properties under Glauber dynamics are discussed.     

Dissolution,Transport and Reaction at a DICY/DGEBA Interface

The curing of an epoxy consisting of the solid hardener dicyandiamide (DICY) and the resin diglycidyl ether of bisphenol A (DGEBA) is studied in a system consisting of a tablet of DICY embedded in liquid DGEBA. Dissolution of DICY within the liquid DGEBA in combination with the transport of dissolved DICY from the tablet border into DGEBA and the chemical reaction of both reactants is studied by scanning Brillouin microscopy and infrared spectroscopy. Scanning Brillouin microscopy demonstrates the spatial and temporal evolution of the static and dynamic hypersonic properties in the course of curing in the vicinity of the DICY tablet. Infrared spectroscopy performed on epoxy pieces extracted from the final sample at different distances from the tablet surface give information about the spatial evolution of the curing process. The results achieved by both techniques are finally combined to yield a better understanding of the curing of DICY-based epoxies, which transform upon curing from strongly heterogeneous systems towards increasingly homogeneous systems.     

Bivalent EGFR-Targeting DARPin-MMAE Conjugates

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5).     

Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

PDZ domains are ubiquitous small protein domains that are mediators of numerous protein–protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling‐transduction complexes. In recent years, PDZ domains have emerged as novel and exciting drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C‐terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys/Arg residue in the ligand‐binding site of the second PDZ domain of PSD‐95, by employing a semisynthetic approach. We generated six semisynthetic PDZ domains comprising different proteogenic and nonproteogenic amino acids representing subtle changes of the conserved Lys/Arg residue. These were tested with four peptide interaction partners, representing the two different binding modes. The results highlight the role of a positively charged amino acid in the β1–β2 loop of PDZ domains, and show subtle differences for canonical and noncanonical interaction partners, thus providing additional insight into the mechanism of PDZ/ligand interaction.     

The influence of hydroelectric power generation on the body composition of juvenile Atlantic salmon

The fat and protein content, and thus energy resources of young (parr) Atlantic salmon (Salmo salar) were examined in a temperate river. Juvenile salmon exhibited distinct seasonal patterns of energy storage and use. The lowest levels of energy were found towards the end of winter (mean mass specific energy about 4.3 kJ·g^?1). There was a rapid accumulation of fat in spring/early summer in spite of low‐water temperatures (highest mean content of mass specific energy of about 5.0 kJ·g^?1 in June). By early autumn (August) there was a rapid depletion of fat and energy resources. The decline in specific energy during late summer was of the same magnitude as the corresponding winter decline. Furthermore, total fat and protein content, and thus energy resources of juveniles below a hydroelectric power plant (hydrostation (HS)) differed from those at a (lower) reference station (RS). The younger fish exhibited higher gains, but also larger losses of energy resources compared to corresponding values at the RS. Specifically during the summer, young of year (YOY) had 31% lower total energy content at the regulated site (mean 1.27 kJ per individual) than the RS (mean 1.84 kJ). Higher mortality may occur at the site most affected by the hydroelectric plant, where energy resources on average were higher, but where depletion was also most severe. Copyright © 2006 John Wiley & Sons, Ltd.     

Presence and level of Campylobacter, coliforms, Escherichia coli, and total aerobic bacteria recovered from broiler parts with and without skin

This study was undertaken to determine if broiler chicken parts without skin are less contaminated with Campylobacter than those with skin. Samples were taken in a commercial plant from defeathered carcasses before evisceration. Bacterial counts from rinse of aseptically removed meat samples were lower than those from stomached skin samples. No Campylobacter were recovered from meat collected from the breasts or thighs, and only 2 of 10 drumstick meat samples had detectable levels of Campylobacter. However, 9 of 10 breast skin, 10 of 10 thigh skin, and 8 of 10 drumstick skin samples were positive for Campylobacter, with between 2 and 3 log10 CFU/g of Campylobacter. Breasts, thighs, and drumsticks were removed from broiler carcasses following evisceration before entering the chill tank. There was a significant difference (50 to 90%) in the levels of Campylobacter on breasts, thighs, and drumsticks with and without skin. Similar trends were noted for coliform, Escherichia coli, and total aerobic bacterial counts from samples collected in the plant. Broiler part samples were also collected at retail outlets. These samples were either skin on and skinned in the laboratory or skin off at purchase. Aseptic removal of skin from broiler breasts, thighs, and drumsticks did not cause change in Campylobacter, coliform, E. coli, or total aerobic counts recovered from the skinned part. Likewise, parts purchased without skin did not have different bacterial counts than paired parts purchased with the skin on. Consumers should not expect to significantly lower the number of bacteria present on a chicken breast, thigh, or drumstick by removing the skin.