Dataset Information

A highly efficient human cell-free translation system.

ABSTRACT: Cell-free protein synthesis (CFPS) systems enable easy in vitro expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently used in vitro systems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor eIF2α. Overexpression of GADD34 and K3L proteins in human cells significantly simplifies cell lysate preparation. The new CFPS system improves the translation of 5' cap-dependent mRNAs as well as those that use internal ribosome entry site (IRES) mediated translation initiation. We find that expression of the GADD34 and K3L accessory proteins before cell lysis maintains low levels of phosphorylation of eIF2α in the extracts. During in vitro translation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. We also find evidence for activation of regulatory pathways related to eukaryotic elongation factor 2 (eEF2) phosphorylation and ribosome quality control in the extracts. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.

SUBMITTER: Aleksashin NA

PROVIDER: S-EPMC9934684 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A highly efficient human cell-free translation system.

Aleksashin Nikolay A NA Chang Stacey Tsai-Lan ST Cate Jamie H D JHD

bioRxiv : the preprint server for biology 20230523

Cell-free protein synthesis (CFPS) systems enable easy <i>in vitro</i> expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently used <i>in vitro</i> systems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor ...[more]

PMID: 36798401

Similar Datasets

Project description:Understanding which factors confer specific functions in any molecular process is fundamental to understanding the system as a whole. Genetic manipulation of cellular systems often leads to the adaptation of gene expression programs, rendering detailed mechanistic insights challenging to isolate and elucidate, particularly in developmental models. The proteome constitutes the ultimate manifestation of gene expression programs; thus, multiple layers of regulation exist to ensure the faithful execution of such programs. While current high-throughput techniques aimed at investigating regulation at the level of translation, such as ribosome profiling and nascent proteomics, can capture nuanced changes in the translational landscape, they suffer from potential biases and confounding factors imposed by adaptation of the cellular states. Cell-free translation systems have been used to elucidate molecular mechanisms for decades, but experimental setups have remained rigid and often required artificially designed mRNA constructs to probe for specialized mechanisms. Here, we detail a human tri-partite cell-free translation system based on the efficient separation of ribosomes, ribosome-depleted cytoplasmic lysate, and mRNAs, allowing for flexible reconstitution of translation reactions. In this setup, some translation components, such as the cytoplasmic lysate, can be kept constant while others, such as ribosome complexes or mRNA complements, can be varied or be subjected to various treatments. We detail several steps and conditions that allow for the translation of complete mRNA populations as input and the detection of nascent peptides from such mRNA populations using mass spectrometry. We utilized this protocol to resolve which components of the translational machinery are selectively affected by environmental and cellular stress conditions that trigger ribosome stalling and collisions, which hitherto have been unresolvable. Finally, we describe how lysates can be prepared from genetically manipulated cells and efficiently and robustly incorporated into high-throughput drug screening pipelines.

Dataset Information

A highly efficient human cell-free translation system.

Publications

A highly efficient human cell-free translation system.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets