Gene expression in a cell-free system on the preparative scale

Baranov, Vladimir; Morozov, Igor Y.; Ortlepp, Stephen A.; Spirin, Alexander S.

doi:10.1016/0378-1119(89)90521-0

Cited by 74 publications

(33 citation statements)

References 14 publications

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“…Studies on the mammalian translation system have led to the idea that its components in vivo are highly organized on the cytoskeleton so as to allow all the intermediates in the process to interact and channel from one to another, without dissociating (53)(54)(55). The initial channeling theory suggested a need for the cellular framework to increase translation efficiency, but using continuous flow cell-free systems, another study showed that tRNAs and translation initiation and elongation factors all remain bound to the protein synthesis machinery and do not exist as freely diffusible molecules (34). Interestingly, this process has been proposed to be the basis for the low efficiency of Sec incorporation in E. coli due to the potential disruption of channeling that must occur during the delivery of SelB by Sec-tRNA Sec (8).…”

Section: Discussionmentioning

confidence: 99%

Regulation of Selenocysteine Incorporation into the Selenium Transport Protein, Selenoprotein P

Shetty

Shah

Copeland

2014

Journal of Biological Chemistry

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Background: Selenocysteine incorporation into selenoprotein P (SEPP1), which contains 10 Sec residues, is unique. Results: Processive Sec incorporation can be reconstituted in vitro, independently of the conserved non-SECIS portions of the 3Ј-UTR. Conclusion: Processivity is intrinsic, but efficiency is governed by selenium levels. Significance: SEPP1 synthesis is essential for male fertility and proper neurologic function.

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Section: Discussionmentioning

confidence: 99%

Regulation of Selenocysteine Incorporation into the Selenium Transport Protein, Selenoprotein P

Shetty

Shah

Copeland

2014

Journal of Biological Chemistry

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“…Spirin et al (7) proposed a continuous flow cell-free translation system, in which a solution containing amino acids and energy sources is supplied to the reaction chamber through a filtration membrane. This design is significantly more efficient than conventional batch systems: The reaction works for tens of hours and produces hundreds of micrograms per milliliter of reaction volume (7)(8)(9). Recently, several modified versions of the Spirin system have been reported (10)(11)(12)(13).…”

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confidence: 99%

A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: Plants apparently contain a suicide system directed at ribosomes

Madin

Sawasaki

Ogasawara

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

436

261

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Current cell-free protein synthesis systems can synthesize proteins with high speed and accuracy, but produce only a low yield because of their instability over time. Here we describe the preparation of a highly efficient but also robust cell-free system from wheat embryos. We first investigated the source of the instability of existing systems in light of endogenous ribosome-inactivating proteins and found that ribosome inactivation by tritin occurs already during extract preparation and continues during incubation for protein synthesis. Therefore, we prepared our system from extensively washed embryos that are devoid of contamination by endosperm, the source of tritin and possibly other inhibitors. In a batch system, we observed continuous translation for 4 h, and sucrose density gradient analysis showed formation of large polysomes, indicating high protein synthesis activity. When the reaction was performed in a dialysis bag, enabling the continuous supply of substrates together with the continuous removal of small byproducts, translation proceeded for >60 h, yielding 1-4 mg of enzymatically active proteins, and 0.6 mg of a 126-kDa tobacco mosaic virus protein, per milliliter of reaction volume. Our results demonstrate that plants contain endogenous inhibitors of translation and that after their elimination the translational apparatus is very stable. This contrasts with the common belief that cell-free translation systems are inherently unstable, even fragile. Our method is useful for the preparation of large amounts of active protein as well as for the study of protein synthesis itself.

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“…However, they exhibit low efficiency, not exceeding few tens of product nanograms in 50 µl reaction. The solution which markedly increased protein expression level involved application of a semipermeable membrane, used for the first time by Spirin (Baranov and Spirin 1993). This produced a significant increase volume of the so-called feeding mix, containing free amino acids, ribonucleotides and high energy molecules (mainly ATP and GTP), securing in parallel high concentration of ribosomes with the yield produced in the reaction compartment (Fig.…”

Section: In Vitro Systems Based On Application Of a Semipermeable Memmentioning

confidence: 99%

The Development of Cell-Free Protein Expression Systems and Their Application in the Research on Antibiotics Targeting Ribosome

Szaflarski¹,

Nowicki²,

Zabel³

2012

Advances in Applied Biotechnology

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Gene expression in a cell-free system on the preparative scale

Cited by 74 publications

References 14 publications

Regulation of Selenocysteine Incorporation into the Selenium Transport Protein, Selenoprotein P

Regulation of Selenocysteine Incorporation into the Selenium Transport Protein, Selenoprotein P

A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: Plants apparently contain a suicide system directed at ribosomes

The Development of Cell-Free Protein Expression Systems and Their Application in the Research on Antibiotics Targeting Ribosome

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