Small changes in energy charge affect protein synthesis in reticulocyte lysates

Rupniak, H. T.; Quincey, R. V.

doi:10.1016/0014-5793(75)80234-1

Cited by 14 publications

(4 citation statements)

References 13 publications

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“…Optimal expression temperatures for other target proteins may therefore vary slightly. Translation initiation and elongation are strongly affected by changes in the ATP/ADP and GTP/GDP ratios (Rupniak and Quincey, ). ATP and GTP concentrations are maintained by an energy regeneration system to ensure increased reaction times.…”

Section: Discussionmentioning

confidence: 99%

Cell‐free protein expression based on extracts from CHO cells

Brödel

Sonnabend

Kubick

2013

Biotech & Bioengineering

127

131

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Protein expression systems are widely used in biotechnology and medicine for the efficient and economic production of therapeutic proteins. Today, cultivated Chinese hamster ovary (CHO) cells are the market dominating mammalian cell-line for the production of complex therapeutic proteins. Despite this outstanding potential of CHO cells, no high-yield cell-free system based on translationally active lysates from these cells has been reported so far. To date, CHO cell extracts have only been used as a foundational research tool for understanding mRNA translation (Lodish et al., 1974;McDowell et al., 1972). In the present study, we address this fact by establishing a novel cell-free protein expression system based on extracts from cultured CHO cells. Lysate preparation, adaptation of in vitro reaction conditions and the construction of particular expression vectors are considered for high-yield protein production. A specific in vitro expression vector, which includes an internal ribosome entry site (IRES) from the intergenic region (IGR) of the Cricket paralysis virus (CrPV), has been constructed in order to obtain optimal performance. The IGR IRES is supposed to bind directly to the eukaryotic 40S ribosomal subunit thereby bypassing the process of translation initiation, which is often a major bottleneck in cell-free systems. The combination of expression vector and optimized CHO cell extracts enables the production of approximately 50 mg/mL active firefly luciferase within 4 h. The batch-type cell-free coupled transcription-translation system has the potential to perform post-translational modifications, as shown by the glycosylation of erythropoietin. Accordingly, the system contains translocationally active endogenous microsomes, enabling the co-translational incorporation of membrane proteins into biological membranes. Hence, the presented in vitro translation system is a powerful tool for the fast and convenient optimization of expression constructs, the specific labeling of integral membrane proteins and the cell-free production of posttranslationally modified proteins.

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Section: Discussionmentioning

confidence: 99%

Cell‐free protein expression based on extracts from CHO cells

Brödel

Sonnabend

Kubick

2013

Biotech & Bioengineering

127

131

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“…These events may be related to altered cerebral energy metabolism. Studies in rabbit reticulocytes indicate that even small changes in energy charge can have significant effects on the rate of protein synthesis (Rupniak and Quincey, 1975). Possibly, a combination of both energy depletion and alterations of ionic homeostasis following activation of NMDA, QUIS, and KAIN receptors by glutamate leads to cell damage.…”

Section: Discussionmentioning

confidence: 99%

Activation of Excitatory Amino Acid Receptors Cannot Alone Account for Anoxia‐Induced Impairment of Protein Synthesis in Rat Hippocampal Slices

1991

Journal of Neurochemistry

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We have investigated the contribution of excitatory amino acid receptor activation to the inhibition of protein synthesis observed after anoxia in rat hippocampal slices. Protein synthesis was assessed in normoxic medium by measuring the incorporation of [14C]lysine into perchloric acid-insoluble tissue extracts. Protein synthesis was impaired after anoxia; the extent of inhibition was dependent on the duration of anoxia and on the time allowed for postanoxic recovery. There was a similar impairment under normoxic conditions when the N-methyl-D-aspartate (NMDA) receptor channel was activated by removing Mg2+ and adding NMDA. This was prevented by noncompetitive antagonists of the NMDA receptor channel (MK-801, phencyclidine, and N-allylnormetazocine). In contrast, incubation with the NMDA antagonists failed to prevent the protein synthesis inhibition caused by anoxia, although it moderately facilitated the postanoxic recovery. Protein synthesis was also impaired under normoxic conditions after incubation with quisqualate and kainate, agonists of non-NMDA glutamate receptors. This impairment was prevented by 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of these receptors. Although 6-cyano-7-nitroquinoxaline-2,3-dione alone failed to prevent anoxic damage, when used in combination with an NMDA antagonist it did partially enhance the later recovery of protein synthesis. These results indicate that the activation of excitatory amino acid receptors cannot alone account for anoxia-induced impairment of protein synthesis in rat hippocampal slices.

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“…1 mutant, which is defective in cytoplasmic mRNA production at the restrictive temperature (11,15). Initiation inhibition, sometimes accompanied by elongation inhibition, has been reported for energy limitation of ascites tumor cells (38,39), thymic lymphocytes (22), reticulocytes (10,19,29), Escherichia coli (8,9,16), and liver (23). It also should be noted that a few reports show inhibition preferentially at an elongation step during energy limitation in liver (3), moss (4), and E. coli (17).…”

Section: Discussionmentioning

confidence: 99%

Regulation of protein synthesis during early limitation of Saccharomyces cerevisiae

Swedes¹,

Dial²,

McLaughlin³

1979

J Bacteriol

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Arsenate, a competitive inhibitor with phosphate in phosphorylation reactions, has been used to lower adenine and guanine nucleotide levels in Saccharomyces cerevisiae to study nucleotide effects on protein synthesis. By measuring polysome levels, we have shown that initiation of protein synthesis is much more sensitive than elongation or termination to inhibition when the ATP/ADP, GTP/ GDP ratios are low. When the arsenate-phosphate molar ratio was 0.27, protein synthesis was inhibited by about 85% and the kinetics of polysome decay was similar to that observed with the initiation inhibitor, verrucarin-76, or with the protein synthesis initiation mutant, ts187, at the restrictive temperature. With this level of arsenate, the adenylate energy charge dropped from 0.9 to 0.7 and the ATP/ADP and GTP/GDP ratios dropped from 6 to 2. The observed correlations between nucleotide ratio changes and inhibition of protein synthesis suggest that the former may be a control signal for the latter. The significance of these in vivo correlations will have to be tested with an in vitro protein synthesizing system. Higher arsenate levels resulted in even lower ATP/ADP, GTP/ GDP ratios and in a slower decay of polysomes, implying that, eventually, elongation (in addition to initiation) was being inhibited.

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Small changes in energy charge affect protein synthesis in reticulocyte lysates

Cited by 14 publications

References 13 publications

Cell‐free protein expression based on extracts from CHO cells

Cell‐free protein expression based on extracts from CHO cells

Activation of Excitatory Amino Acid Receptors Cannot Alone Account for Anoxia‐Induced Impairment of Protein Synthesis in Rat Hippocampal Slices

Regulation of protein synthesis during early limitation of Saccharomyces cerevisiae

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