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Functional analysis of membrane proteins produced by cell-free translation

: Dondapati, S.K.; Wüstenhagen, D.A.; Kubick, S.

Fulltext ()

Bornscheuer, U.T.:
Protein Engineering. Methods and Protocols
New York/NY: Humana Press, 2018 (Methods in molecular biology 1685)
ISBN: 978-1-4939-7364-4 (Print)
ISBN: 978-1-4939-7366-8 (Online)
ISBN: 1-4939-7364-9
Book Article, Electronic Publication
Fraunhofer IZI ()

Cell-free production is a valuable and alternative method for the synthesis of membrane proteins. This system offers openness allowing the researchers to modify the reaction conditions without any boundaries. Additionally, the cell-free reactions are scalable from 20 μL up to several mL, faster and suitable for the high-throughput protein production. Here, we present two cell-free systems derived from Escherichia coli (E. coli) and Spodoptera frugiperda (Sf21) lysates. In the case of the E. coli cell-free system, nanodiscs are used for the solubilization and purification of membrane proteins. In the case of the Sf21 system, endogenous microsomes with an active translocon complex are present within the lysates which facilitate the incorporation of the bacterial potassium channel KcsA within the microsomal membranes. Following cell-free synthesis, these microsomes are directly used for the functional analysis of membrane proteins.