Cell‐free production of active <i>E. coli</i> thioredoxin reductase and glutathione reductase

Knapp, Kurtis G.; Swartz, James R.

doi:10.1016/s0014-5793(04)00025-0

Cited by 19 publications

(19 citation statements)

References 18 publications

(23 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This step is essential for the recycling of these thiol-containing molecules so that they can be functional in the thioredoxin-thioredoxin reductase and glutathione-glutathione reductase systems, which maintain the reduced state of the cytoplasm (58). Previous research indicates that the activities of FDR depend on the FAD concentration during cofactor incorporation (59). In this study, the low FAD content at the high temperature could result in a less-active FDR, which causes the oxidative stress.…”

Section: Discussionmentioning

confidence: 75%

Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin

Chen

Shen

Solem

et al. 2013

Appl Environ Microbiol

View full text Add to dashboard Cite

Lactococcus lactis MG1363 was found to be unable to grow at temperatures above 37°C in a defined medium without riboflavin, and the cause was identified to be dissolved oxygen introduced during preparation of the medium. At 30°C, growth was unaffected by dissolved oxygen and oxygen was consumed quickly. Raising the temperature to 37°C resulted in severe growth inhibition and only slow removal of dissolved oxygen. Under these conditions, an abnormally low intracellular ratio of [ATP] to [ADP] (1.4) was found (normally around 5), which indicates that the cells are energy limited. By adding riboflavin to the medium, it was possible to improve growth and oxygen consumption at 37°C, and this also normalized the [ATP]-to-[ADP] ratio. A codon-optimized redox-sensitive green fluorescent protein (GFP) was introduced into L. lactis and revealed a more oxidized cytoplasm at 37°C than at 30°C. These results indicate that L. lactis suffers from heat-induced oxidative stress at increased temperatures. A decrease in intracellular flavin adenine dinucleotide (FAD), which is derived from riboflavin, was observed with increasing growth temperature, but the presence of riboflavin made the decrease smaller. The drop was accompanied by a decrease in NADH oxidase and pyruvate dehydrogenase activities, both of which depend on FAD as a cofactor. By overexpressing the riboflavin transporter, it was possible to improve FAD biosynthesis, which resulted in increased NADH oxidase and pyruvate dehydrogenase activities and improved fitness at high temperatures in the presence of oxygen. Lactococcus lactis is a mesophilic bacterium with an optimum growth temperature around 30°C (1) that is widely used in the dairy industry for production of cheese and buttermilk, where it plays a crucial role in flavor and texture formation. During cheese production, L. lactis is frequently exposed to a variety of stresses, such as heat stress (2). Superoptimal temperatures cause the denaturation of macromolecules and induce chaperone proteins, such as DnaK, GroEL, and GroES, which then help proteins to fold correctly (2). The chaperones are essential for growth at elevated temperatures, and mutants with defects in chaperone activity display a pronounced temperature-sensitive phenotype (3). The opposite effect is sometimes seen when overexpressing chaperones, and in L. lactis this results in improved fitness and lactic acid production at high temperatures (4, 5). The viability of some L. lactis strains at high temperatures is also improved by an incorporation of high concentrations of NaCl into the medium or a preadaptation prior to inoculation (6), and this phenomenon was demonstrated to be correlated with an induction of several chaperones (7). Meanwhile, metabolic responses are regarded as important factors in handling heat stress. It has been demonstrated that genes in several metabolic pathways are important for handling heat stress (8), and a recently recognized c-di-AMP-specific phosphodiesterase in L. lactis was also found to be important for heat tole...

show abstract

Section: Discussionmentioning

confidence: 75%

Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin

Chen

Shen

Solem

et al. 2013

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…We expect that similar modifications to cell-free systems will have direct applications for the study of other metalloproteins, oxygen-sensitive proteins, and proteins requiring helpers to mature. Additionally, the cell-free system has proven to be adaptable for the production of mammalian proteins (Yang et al, 2005), proteins requiring disulfide bonds (Yin and Swartz, 2004), proteins requiring multiple subunits, proteins requiring [Fe-S] centers and other cofactors (Boyer et al, 2006;Knapp and Swartz, 2004), membrane proteins, proteome libraries, and proteins requiring chaperones (Yin and Swartz, 2004). In combination, these capabilities establish this system as a highly adaptable platform for the production and study of many complex proteins.…”

Section: Resultsmentioning

confidence: 99%

“…Cell extracts have been improved through strain engineering and modified preparation procedures to enhance the cellfree production of many types of proteins (Boyer et al, 2006;Knapp and Swartz, 2004;Miyazaki-Imamura et al, 2003;Yang et al, 2004;Yin and Swartz, 2004). Two unprecedented adaptations in extract preparation were implemented for expression of active hydrogenase proteins: the inclusion of heterologous maturation enzymes, and the preparation of extracts under strictly anaerobic conditions.…”

Section: Optimization Of Cell Extracts For Cell-free Expression Of [Fmentioning

confidence: 99%

“…This allows facile addition of substrates, even to concentrations unachievable in vivo. The ability to control the expression environment has enabled production of proteins requiring [Fe-S] clusters (Boyer et al, 2006), FAD cofactors (Knapp and Swartz, 2004), heme groups (Miyazaki-Imamura et al, 2003), and disulfide bonds (Yin and Swartz, 2004). Since modern cell-free systems more closely mimic the natural cytoplasmic folding environment of cells (Jewett and Swartz, 2004), protein folding can occur co-translationally, and activation may be assisted by intact pathways from E. coli.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cell‐free synthesis and maturation of [FeFe] hydrogenases

Boyer

Stapleton

Kuchenreuther

et al. 2007

Biotech & Bioengineering

105

View full text Add to dashboard Cite

[FeFe] hydrogenases catalyze the reversible reduction of protons to molecular hydrogen (Adams (1990); Biochim Biophys Acta 1020(2): 115-145) and are of significant interest for the biological production of hydrogen fuel. They are complex proteins with active sites containing iron, sulfur, and carbon monoxide and cyanide ligands (Peters et al. (1998); Science 282(5395): 1853-1858). Maturation enzymes for [FeFe] hydrogenases have been identified (Posewitz et al. (2004); J Biol Chem 279(24): 25711-25720), but complete mechanisms have not yet been elucidated. The study of [FeFe] hydrogenases has been impeded by the lack of an easily manipulated expression/activation system capable of producing these complex and extremely oxygen-sensitive enzymes. Here we show the first expression of functional [FeFe] hydrogenases in an Escherichia coli-based cell-free transcription/translation system. We have produced and matured both algal and bacterial hydrogenases using E. coli cell extracts containing the HydG, HydE, and HydF proteins from Shewanella oneidensis. The current system produces approximately 22 microg/mL of active protein, constituting approximately 44% of the total protein produced. Active protein yield is greatly enhanced by pre-incubation of the maturation enzyme-containing extract with inorganic iron and sulfur for reconstitution of the [Fe-S] clusters in HydG, HydE, and HydF. The absence of cell walls permits direct addition of cofactors and substrates, enabling rapid production of active protein and providing control over the maturation conditions. These new capabilities will enhance the investigation of complex proteins requiring helper proteins for maturation and move us closer to the development of improved hydrogenases for biological production of hydrogen as a clean, renewable alternative fuel.

show abstract

“…Thioredoxin reductase was produced in CFPS reactions as described previously (Knapp and Swartz, 2004). The pHs of aliquots of the unpurified reaction product were adjusted to the desired values with small volumes of concentrated KOH or HCl.…”

Section: Assay For Thioredoxin Reductase Activitymentioning

confidence: 99%

Cell‐free synthesis of proteins that require disulfide bonds using glucose as an energy source

Knapp

Goerke

Swartz

2007

Biotech & Bioengineering

View full text Add to dashboard Cite

The primary objective of this work was to create a cell-free protein synthesis extract that produces proteins requiring disulfide bonds while using glucose as an energy source. We attempted to avoid using iodoacetamide (IAM) to stabilize the required oxidizing thiol redox potential, since previous IAM pretreatments prevented glucose utilization apparently by inactivating glyceraldehyde 3-phosphate dehydrogenase (G-3PDH). Instead, the glutathione reductase (Gor)-mediated disulfide reductase system was disabled by deleting the gor gene from the KC6 cell-extract source strain. The thioredoxin reductase (TrxB)-mediated system was disabled by first adding a purification tag to the trxB gene in the chromosome to create strain KGK10 and then by affinity removal of the tagged TrxB. This was expected to result in a cell extract devoid of all disulfide reductase activity, but this was not the case. Although the concentration of IAM required to stabilize oxidized glutathione in the KGK10 extract could be reduced 20-fold, IAM pretreatment was still required to avoid disulfide reduction. Nonetheless, active urokinase and murine granulocyte macrophage-colony stimulating factor (mGM-CSF) were produced in reactions with KGK10 extract either with affinity removal of TrxB or with 50 microM IAM pretreatment. With the less intensive IAM pretreatment, glucose could be used as an energy source in a production system that promotes oxidative protein folding. This new protocol offers an economically feasible cell-free system for the production of secreted mammalian proteins as human therapeutics or vaccines.

show abstract

Cell‐free production of active E. coli thioredoxin reductase and glutathione reductase

Cited by 19 publications

References 18 publications

Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin

Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin

Cell‐free synthesis and maturation of [FeFe] hydrogenases

Cell‐free synthesis of proteins that require disulfide bonds using glucose as an energy source

Contact Info

Product

Resources

About