Project Details
Projekt
Development of processes for the production of the signaling factors BMP-2, TGF-ß3, Smad8 L MH2 as well as novel variants with defined stabilities
Subject Area
Biomaterials
Term
from 2015 to 2022
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 251503496
Subproject 2 deals with the development of processes for the production of those signaling factors which are relevant for the research consortium, namely BMP-2, TGF-beta3 and Smad8 L MH2 as well as additional variants with defined functionalities (e.g. fluorescence tags for in vitro and in vivo monitoring, tags for an improved binding to the extracellular matrix). For the production of the structurally related cystine knot proteins BMP-2 and TGF-beta3 expression systems as well as production processes will be developed which allow the generation of bioactive proteins using either bacterial, or if required, also mammalian expression systems. Moreover, novel, preferentially bacterial, expression systems and production processes for the generation of the signaling Smad proteins will be generated. For the purification of these signaling factors, novel, membrane-adsorber based purification processes will be developed to allow easy and selective target protein purification from complex protein mixtures. The optimization of production and purification procedures will be first applied to the generation of soluble unmodified signal proteins. Furthermore, research activities will be devoted to target the signal proteins, in particular the Smad proteins, into bacterial inclusion bodies where they should deposit as biologically active proteins in a framework of amyloidic variants of the target protein. Signal proteins produced this way will be tested for their applicability as functional protein depots useful for the controllable release in a time and position dependent manner. Moreover, variants of the signal proteins with defined functionalities (e.g. fluorescence tagging, tagging for improved binding to the extracellular matrix) by utilizing point mutations or fusion tags will be developed and tested for graded release. For tagging also novel possibilities will be investigated to visualize cytokines at their site of action. For this small, selective binders (aptamers) to cytokines will be tested. They will be labeled directly or later. For cytokine production in mammalian expression systems, transient transfection systems (employing CHO or HEK cells) will be established using microfluidic systems.
DFG Programme
Research Units
