Development, validation, and application of the ribosome separation and reconstitution system for protein translation in vitro

Trainor, Brandon M; Pestov, Dimitri G.; Shcherbik, Natalia

doi:10.1261/rna.078852.121

Cited by 2 publications

(3 citation statements)

References 51 publications

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“…Trainor et al describe a protocol for separating the ribosomes from the rest of the cell-free extract in yeast 45 . We therefore adopted the protocol of Trainor et al with modi cations to the HEK293 extract and tested the time-inhibition in the resulting fractions.…”

Section: Translationally Active Fraction Of Hek293 Extractmentioning

confidence: 99%

“…Cells were lysed as described above. The ribosome free extract and pellet fractions were prepared according to a previously published procedure 45 . The cell lysate was ultracentrifuged with the Beckman Optima MAX-XP in an MLA-150 rotor at 180 000 × g for 2 h (or 20 min) at 4 o C. The supernatant (R-dep) was aliquoted in 50 µl volumes, ashfrozen in liquid nitrogen and stored at -80 o C.…”

Section: Declarationsmentioning

confidence: 99%

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Unraveling time-inhibition mechanisms in mammalian cell-free protein synthesis

Mansour,

Kipper,

Pulk

2024

Preprint

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We present a highly efficient human HEK293-based cell-free in vitro protein synthesis (CFPS) system that produces 300 µg/ml of reporter protein. The main challenge with the CFPS assay is its limited activity for a few hours, during which all protein is synthesized. If the activity of the CFPS system can be extended, more protein can be produced. The time-dependent inhibition has been studied in the yeast system, but not much is known in the mammalian system. We used the HEK293 CFPS assay to investigate the reasons for time inhibition. We observed that the main culprit is the energy regeneration system, which is depleted quickly. We also demonstrate that the CFPS assay can be used with other mammalian cells or tissues, as evidenced by the active human neuroblastoma SH-SY5Y-based CFPS assay. We observe differences between the yeast and mammalian systems; for example, there is no need to add creatine kinase (CK) as the native CK is functional. This knowledge helps to reduce the costs of CFPS-based systems for biotechnological purposes.

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Section: Translationally Active Fraction Of Hek293 Extractmentioning

confidence: 99%

Section: Declarationsmentioning

confidence: 99%

Unraveling time-inhibition mechanisms in mammalian cell-free protein synthesis

Mansour,

Kipper,

Pulk

2024

Preprint

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“…A technique termed the hybrid translation system , which is based on the exchange of ribosomes, is an alternative option in such studies ( Panthu et al, 2015 ; Penzo et al, 2016 ; Erales et al, 2017 ; Trainor et al, 2021 ). Given that ultracentrifugation sediments the ribosome into the pellet, RRL can be prepared as ribosome-free but translation-competent only when the ribosomes are replenished.…”

Section: Introductionmentioning

confidence: 99%

Human-rabbit Hybrid Translation System to Explore the Function of Modified Ribosomes

Matsuura-Suzuki¹,

Toh²,

Iwasaki³

2023

BIO-PROTOCOL

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In vitro translation systems are a useful biochemical tool to research translational regulation. Although the preparation of translation-competent cell extracts from mammals has often been a challenge, the commercially available rabbit reticulocyte lysate (RRL) is an exception. However, its valid use, investigating the mechanism of translation machinery such as ribosomes in RRL, presents an analytic hurdle. To overcome this issue, the hybrid translation system, which is based on the supplementation of purified human ribosomes into ribosome-depleted RRL, has been developed. Here, we describe the step-by-step protocol of this system to study translation driven by ribosomes lacking post-translational modifications of the ribosomal protein. Moreover, we combined this approach with a previously developed reporter mRNA to assess the processivity of translation elongation. This protocol could be used to study the potency of heterologous ribosomes.

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Development, validation, and application of the ribosome separation and reconstitution system for protein translation in vitro

Cited by 2 publications

References 51 publications

Unraveling time-inhibition mechanisms in mammalian cell-free protein synthesis

Unraveling time-inhibition mechanisms in mammalian cell-free protein synthesis

Human-rabbit Hybrid Translation System to Explore the Function of Modified Ribosomes

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