Author: Gurzeler, Lukas-Adrian; Ziegelmüller, Jana; Mühlemann, Oliver; Karousis, Evangelos D.
Title: Production of human translation-competent lysates using dual centrifugation Cord-id: afgghhdw Document date: 2021_7_30
ID: afgghhdw
Snippet: Protein synthesis is a central process in gene expression and the development of efficient in vitro translation systems has been the focus of scientific efforts for decades. The production of translation-competent lysates originating from human cells or tissues remains challenging, mainly due to the variability of cell lysis conditions. Here we present a robust and fast method based on dual centrifugation that allows for detergent-free cell lysis under controlled mechanical forces. We optimized
Document: Protein synthesis is a central process in gene expression and the development of efficient in vitro translation systems has been the focus of scientific efforts for decades. The production of translation-competent lysates originating from human cells or tissues remains challenging, mainly due to the variability of cell lysis conditions. Here we present a robust and fast method based on dual centrifugation that allows for detergent-free cell lysis under controlled mechanical forces. We optimized the lysate preparation to yield cytoplasmic extracts from human cells that efficiently translate mRNAs in a cap-dependent as wells as in an IRES-mediated way. Reduction of the phosphorylation state of eIF2α using recombinant GADD34 and 2-aminopurine considerably boosts the protein output, reinforcing the potential of this method for the production of recombinant proteins from human lysates.
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