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Production of human translation-competent lysates using dual centrifugation.

ABSTRACT: 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 cytoplasm-enriched extracts from human cells that efficiently translate mRNAs in a cap-dependent as well 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 to produce recombinant proteins from human lysates.

SUBMITTER: Gurzeler LA

PROVIDER: S-EPMC8815625 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Production of human translation-competent lysates using dual centrifugation.

Gurzeler Lukas-Adrian LA Ziegelmüller Jana J Mühlemann Oliver O Karousis Evangelos D ED

RNA biology 20211229 1

Protein synthesis is a central process in gene expression and the development of efficient <i>in vitro</i> 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 opt ...[more]

PMID: 34965175

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Production of human translation-competent lysates using dual centrifugation.

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Production of human translation-competent lysates using dual centrifugation.

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