idw – Informationsdienst Wissenschaft
Nachrichten, Termine, Experten
Nachrichten, Termine, Experten
Abstract: Membrane proteins have become an important focus of the current efforts in structural and functional genomics and the rapid progress of various genome sequencing projects has greatly accelerated the discovery of novel genes encoding membrane proteins. In contrast, the molecular analysis of membrane proteins lags far behind that of cytosolic soluble proteins. Preparing high quality samples of functionally folded proteins represents a major bottleneck that restricts further structural and functional studies. Cell-free protein expression systems, in particular those of eukaryotic origin, have recently been developed as promising tools for the rapid and efficient production of a wide variety of membrane proteins. A large number of these proteins, however, require posttranslational modifications for optimum function. Several membrane proteins have been expressed in vivo to date, most of them being functionally, antigenically, and immunogenically similar to their authentic counterparts. This is mainly due to the properties of cultured eukaryotic cells, which are able to carry out many types of posttranslational modifications such as the addition of N- and O- linked oligosaccharides, but also palmitoylation, myristylation, and phosphorylation. Based on these versatile properties of cultured cell lines, we have developed a technique for the standardized production of translationally active eukaryotic lysates from insect cells. In contrast to other cell-free protein synthesis systems (e.g., rabbit reticulocyte lysates and wheat germ extracts) our homogenization procedure avoids any serious breakdown of membrane vesicles already existing in the cytoplasm of the prepared eukaryotic cells. We have demonstrated the functional integrity of these subcellular components by showing signal peptide cleavage as well as glycosylation of in vitro expressed membrane proteins. Moreover, we have expanded our cell-free protein expression system by the insertion of orthogonal tRNA/synthetase pairs to facilitate the cotranslational and site directed incorporation of non-canonical building blocks. These fluorescently labeled and chemoselective moieties enable the site-specific modification of de novo synthesized membrane proteins.
The development of this novel eukaryotic in vitro translation system now expands the possibilities of cell-free protein synthesis, since posttranslational modifications significantly alter the physical and chemical properties of membrane proteins, including their folding and conformational distribution and these modifications are frequently a fundamental prerequisite for functional activity.
Information on participating / attending:
Einladender: Prof. Dr. Uwe Bornscheuer
Kontakt: PD Dr. Heike Kahlert, Vorsitzende des Ortsverbandes der GDCh
hkahlert@uni-greifswald.de
Date:
05/18/2015 16:15 - 05/18/2015 17:15
Event venue:
Institut für Biochemie, Großer Hörsaal
17489 Greifswald
Mecklenburg-Vorpommern
Germany
Target group:
Scientists and scholars, Students
Relevance:
transregional, national
Subject areas:
Chemistry
Types of events:
Presentation / colloquium / lecture
Entry:
02/16/2015
Sender/author:
Sabine Köditz
Department:
Presse- und Informationsstelle
Event is free:
no
Language of the text:
German
URL of this event: http://idw-online.de/en/event50013
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