Artificial environments for the co-translational stabilization of cell-free expressed proteins

An approach for designing individual expression environments that reduce or prevent protein aggregation and precipitation is described. Inefficient folding of difficult proteins in unfavorable translation environments can cause significant losses of overexpressed proteins as precipitates or inclusion bodies. A number of chemical chaperones including alcohols, polyols, polyions or polymers are known to have positive effects on protein stability. However, conventional expression approaches can use such stabilizing agents only post-translationally during protein extraction and purification. Proteins that already precipitate inside of the producer cells cannot be addressed. The open nature of cell-free protein expression systems offers the option to include single chemicals or cocktails of stabilizing compounds already into the expression environment. We report an approach for systematic screening of stabilizers in order to improve the solubility and quality of overexpressed proteins co-translationally. A comprehensive list of representative protein stabilizers from the major groups of naturally occurring chemical chaperones has been analyzed and their concentration ranges tolerated by cell-free expression systems have been determined. As a proof of concept, we have applied the method to improve the yield of proteins showing instability and partial precipitation during cell-free synthesis. Stabilizers that co-translationally improve the solubility and functional folding of human glucosamine 6-phosphate N-acetyltransferase have been identified and cumulative effects of stabilizers have been studied

Modellierung einer wissensbasierten, interaktiven Prozeßsteuerung

Der zunehmende Bedarf, wissensbasierte Systeme für realistische Anwendungen einzusetzen, stellt eine Herausforderung an die Modellierung der zugrundeliegenden Domäne dar. Einführungen zu Expertensystemen, Wissensrepräsentation sowie Wissensverarbeitung findet man beispielsweise in [Pup91], [Str91], [Bib93], [Hen91], [Pup90], [BJT96b], [Sch98] und [Min74]. Einige kritische Betrachtungen zu Fragen der Wissensverarbeitung findet man in [BHJR94] oder auch in [GS96]. Speziell auf die Problematik der Wissensrepräsentation in komplexen Domänen wird in [Hel96] eingegangen. Um neben dem theoretischen Wissen auch praktische Erfahrungen bei der Modellierung von Anwendungsfällen zu erlangen, wurde mit einigen Studenten des Studiengangs Informatik ein entsprechendes Praxisseminar durchgeführt. In diesem sollten sie ihr bereits in den entsprechenden Lehrveranstaltungen erworbenes theoretisches Wissen zu Expertensystemen, Wissensrepräsentation, Wissensverarbeitung und Planung vertiefen und praktisch anwenden

Schwerpunkte bei der Wissensmodellierung am Beispiel einer Prozeßsimulation

Der zunehmende Bedarf, wissensbasierte Systeme für realistische Anwendungen einzusetzen, stellt eine Herausforderung an die Modellierung der zugrunde liegenden Domäne dar. Bei wichtigen Entscheidungen wird dem Entwerfer zur Zeit nur ungenügende oder keine Unterstützung seitens der Entwurfssysteme gegeben. Um beim Systementwurf Erfahrungen zu sammeln, wurde mit Studenten ein Praxisseminar durchgeführt. Die dort gemachten Erfahrungen bezüglich der Bedeutung verschiedener Entwurfsentscheidungen werden nachfolgend beschrieben

ER Targeting and Insertion of Tail-Anchored Membrane Proteins by the GET Pathway

Hundreds of eukaryotic membrane proteins are anchored to membranes by a single transmembrane domain at their C-terminus. Many of these tail-anchored (TA) proteins are post-translationally targeted to the endoplasmic reticulum (ER) membrane for insertion by the Guided-Entry of TA protein insertion (GET) pathway. In recent years most of the components of this conserved pathway have been biochemically and structurally characterized. Get3 is the pathway targeting factor that utilizes nucleotide-linked conformational changes to mediate the delivery of TA proteins between the GET pre-targeting machinery in the cytosol and the transmembrane pathway components in the ER. Here we focus on the mechanism of the yeast GET pathway and make a speculative analogy between its membrane insertion step and the ATPase-driven cycle of ABC transporters.Molecular and Cellular Biolog

Membrane protein production in Escherichia coli cell-free lysates

AbstractCell-free protein production has become a core technology in the rapidly spreading field of synthetic biology. In particular the synthesis of membrane proteins, highly problematic proteins in conventional cellular production systems, is an ideal application for cell-free expression. A large variety of artificial as well as natural environments for the optimal co-translational folding and stabilization of membrane proteins can rationally be designed. The high success rate of cell-free membrane protein production allows to focus on individually selected targets and to modulate their functional and structural properties with appropriate supplements. The efficiency and robustness of lysates from Escherichia coli strains allow a wide diversity of applications and we summarize current strategies for the successful production of high quality membrane protein samples

Requirements on Paramagnetic Relaxation Enhancement Data for Membrane Protein Structure Determination by NMR

SummaryNuclear magnetic resonance (NMR) structure calculations of the α-helical integral membrane proteins DsbB, GlpG, and halorhodopsin show that distance restraints from paramagnetic relaxation enhancement (PRE) can provide sufficient structural information to determine their structure with an accuracy of about 1.5 Å in the absence of other long-range conformational restraints. Our systematic study with simulated NMR data shows that about one spin label per transmembrane helix is necessary for obtaining enough PRE distance restraints to exclude wrong topologies, such as pseudo mirror images, if only limited other NMR restraints are available. Consequently, an experimentally realistic amount of PRE data enables α-helical membrane protein structure determinations that would not be feasible with the very limited amount of conventional NOESY data normally available for these systems. These findings are in line with our recent first de novo NMR structure determination of a heptahelical integral membrane protein, proteorhodopsin, that relied extensively on PRE data

Das Konzept der Therapievollständigkeit medizinischer Wissensbasen

Das Stellen von Diagnosen ist ein in verschiedenen Gebieten in ähnlicher Form wiederkehrendes Problem. Ziel ist es, unter Nutzung von Beobachtungen des fehlerhaften Systems die Ursachen für das aktuelle Fehlverhalten zu ermitteln. In den bisher vorliegenden Theorien der modellbasierten Diagnose wird die Therapie als beeinflussender Faktor vernachlässigt und als externes (nachfolgendes) Problem betrachtet. Da diagnostische Expertensysteme in der Medizin aber den jeweiligen Untersuchungs- und Behandlungsmöglichkeiten des Arztes einer bestimmten Spezialisierungstiefe entsprechen sollten, scheint es angebracht, in eine Theorie der Diagnose auch das Konzept der Therapie einzubringen

The C-terminus of p63 contains multiple regulatory elements with different functions

The transcription factor p63 is expressed as at least six different isoforms, of which two have been assigned critical biological roles within ectodermal development and skin stem cell biology on the one hand and supervision of the genetic stability of oocytes on the other hand. These two isoforms contain a C-terminal inhibitory domain that negatively regulates their transcriptional activity. This inhibitory domain contains two individual components: one that uses an internal binding mechanism to interact with and mask the transactivation domain and one that is based on sumoylation. We have carried out an extensive alanine scanning study to identify critical regions within the inhibitory domain. These experiments show that a stretch of ~13 amino acids is crucial for the binding function. Further, investigation of transcriptional activity and the intracellular level of mutants that cannot be sumoylated suggests that sumoylation reduces the concentration of p63. We therefore propose that the inhibitory function of the C-terminal domain is in part due to direct inhibition of the transcriptional activity of the protein and in part due to indirect inhibition by controlling the concentration of p63. Keywords: p63, transcriptional regulation, auto-inhibition, sumoylatio

The parallel G-quadruplex structure of vertebrate telomeric repeat sequences is not the preferred folding topology under physiological conditions

G-quadruplex topologies of telomeric repeat sequences from vertebrates were investigated in the presence of molecular crowding (MC) mimetics, namely polyethylene glycol 200 (PEG), Ficoll 70 as well as Xenopus laevis egg extract by CD and NMR spectroscopy and native PAGE. Here, we show that the conformational behavior of the telomeric repeats in X. laevis egg extract or in Ficoll is notably different from that observed in the presence of PEG. While the behavior of the telomeric repeat in X. laevis egg extract or in Ficoll resembles results obtained under dilute conditions, PEG promotes the formation of high-order parallel topologies. Our data suggest that PEG should not be used as a MC mimetic