• Slide 1
  • KcsA

    Representative current trace recorded from a planar lipid bilayer in which purified KcsA channels were reconstituted (measured on the Port-a-Patch)

2018 - Functional Analysis of Membrane Proteins Produced by Cell-Free Translation

icon pap   Port-a-Patch and   Icon Orbit Mini   Orbit mini article in Protein Engineering (2018)

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

Book chapter:
In: Protein Engineering (2018), chapter: "Functional Analysis of Membrane Proteins Produced by Cell-Free Translation" pp 171-186


Abstract:

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.


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