scholarly journals Optimization of an In Vitro Transcription/Translation System Based on Sulfolobus solfataricus Cell Lysate

Archaea ◽  
2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Giada Lo Gullo ◽  
Rosanna Mattossovich ◽  
Giuseppe Perugino ◽  
Anna La Teana ◽  
Paola Londei ◽  
...  
Keyword(s):  
High Efficiency ◽  
Specific Activity ◽  
Expression System ◽  
Sulfolobus Solfataricus ◽  
Essential Element ◽  
23S Rrna ◽  
Translation System ◽  
Cell Lysate ◽  
Cell Extracts

A system is described which permits the efficient synthesis of proteins in vitro at high temperature. It is based on the use of an unfractionated cell lysate (S30) from Sulfolobus solfataricus previously well characterized in our laboratory for translation of pretranscribed mRNAs, and now adapted to perform coupled transcription and translation. The essential element in this expression system is a strong promoter derived from the S. solfataricus 16S/23S rRNA-encoding gene, from which specific mRNAs may be transcribed with high efficiency. The synthesis of two different proteins is reported, including the S. solfataricus DNA-alkylguanine-DNA-alkyl-transferase protein (SsOGT), which is shown to be successfully labeled with appropriate fluorescent substrates and visualized in cell extracts. The simplicity of the experimental procedure and specific activity of the proteins offer a number of possibilities for the study of structure-function relationships of proteins.

scholarly journals The Herpes Simplex Virus vhs Protein Induces Endoribonucleolytic Cleavage of Target RNAs in Cell Extracts

1999 ◽  
Vol 73 (9) ◽  
pp. 7153-7164 ◽  
Author(s):  
Mabrouk M. Elgadi ◽  
Christopher E. Hayes ◽  
James R. Smiley
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Pseudorabies Virus ◽  
Host Protein ◽  
In Vitro Translation ◽  
Rna Decay ◽  
Translation System ◽  
Cell Extracts ◽  
Simplex Virus

ABSTRACT The herpes simplex virus virion host shutoff (vhs) protein (UL41 gene product) is a component of the HSV virion tegument that triggers shutoff of host protein synthesis and accelerated mRNA degradation during the early stages of HSV infection. Previous studies have demonstrated that extracts from HSV-infected cells and partially purified HSV virions display vhs-dependent RNase activity and that vhs is sufficient to trigger accelerated RNA degradation when expressed as the only HSV protein in an in vitro translation system derived from rabbit reticulocytes. We have used the rabbit reticulocyte translation system to characterize the mode of vhs-induced RNA decay in more detail. We report here that vhs-dependent RNA decay proceeds through endoribonucleolytic cleavage, is not affected by the presence of a 5′ cap or a 3′ poly(A) tail in the RNA substrate, requires Mg2+, and occurs in the absence of ribosomes. Intriguingly, sites of preferential initial cleavage were clustered over the 5′ quadrant of one RNA substrate that was characterized in detail. The vhs homologue of pseudorabies virus also induced accelerated RNA decay in this in vitro system.

Electron microscope autoradiography of isolated molecules

1978 ◽  
Vol 36 (2) ◽  
pp. 192-193
Author(s):  
M. Bouteille ◽  
E. Delain ◽  
N. Angelier
Keyword(s):  
Electron Microscope ◽  
High Efficiency ◽  
Specific Activity ◽  
Molecular Complexes ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
Loop Technique ◽  
Silver Grains ◽  
Isolated Molecules

The LIGOP method of electron microscope autoradiography which consists in a combination of coating Ilford emulsion with the loop technique and developing with gold latensification and phenidon has proved to provide small, compact developed silver grains with high efficiency.This has made it possible to use this technique with very small materials such as isolated molecules of molecular complexes.The method was assayed first with 3H-Thymidine labelled T7 phages DNA molecule with 630,000 cpm/μg specific activity (fig. 1). The molecules were spread using the adsorption technique constrasted by rotatory shadowing with platinum and then subjected to autoradiography. The Labelling was sufficient to obtain quantitative data in which the spread molecules were considered as a material comparable to a “hot line”. The efficiency (45%) and the HD value (1600 Å) were calculated.The method was also applied to transcription units of pleurodeles oocytes nucleoli (fig. 2) labelled in vitro with 3H-Uridine.

scholarly journals In Vitro Processing of the 16S rRNA of the Thermophilic Archaeon Sulfolobus solfataricus

2001 ◽  
Vol 183 (13) ◽  
pp. 3866-3874 ◽  
Author(s):  
Andrea Ciammaruconi ◽  
Paola Londei
Keyword(s):  
16S Rrna ◽  
Sulfolobus Solfataricus ◽  
Conserved Sequence ◽  
Processing Enzymes ◽  
Cell Extracts ◽  
In Vitro Processing ◽  
16S Rna ◽  
Thermophilic Archaeon ◽  
Rna Maturation

ABSTRACT In this paper we have analyzed the processing in vitro of the 16S rRNA of the thermophilic archaeon Sulfolobus solfataricus, using pre-rRNA substrates transcribed in vitro and different protein preparations as the source of processing enzymes. We show that the 5′ external transcribed spacer of the S. solfataricus pre-rRNA transcript contains a target site for a specific endonuclease, which recognizes a conserved sequence also existing in the early A0 and 0 processing sites of Saccharomyces cerevisiae and vertebrates. This site is present in other members of the kingdomCrenarchaeota but apparently not in theEuryarchaeota. Furthermore, S. solfataricuspre-16S RNA is processed within the double-helical stem formed by the inverted repeats flanking the 16S RNA sequence, in correspondence with a bulge-helix-bulge motif. The endonuclease responsible for this cleavage is present in both the Crenarchaeota and theEuryarchaeota. The processing pattern remained the same when the substrate was a 30S ribonucleoprotein particle instead of the naked RNA. Maturation of either the 5′ or the 3′ end of the 16S RNA molecule was not observed, suggesting either that maturation requires conditions not easily reproducible in vitro or that the responsible endonucleases are scarcely represented in cell extracts.

scholarly journals Mercury Inactivates Transcription and the Generalized Transcription Factor TFB in the Archaeon Sulfolobus solfataricus

2004 ◽  
Vol 48 (6) ◽  
pp. 1993-1999 ◽  
Author(s):  
Vidula Dixit ◽  
Elisabetta Bini ◽  
Melissa Drozda ◽  
Paul Blum
Keyword(s):  
Transcription Factor ◽  
Mercuric Chloride ◽  
Sulfolobus Solfataricus ◽  
In Vitro Transcription ◽  
Mercury Toxicity ◽  
Transcription Activity ◽  
Treated Cell ◽  
Mercuric Ion ◽  
Cell Extracts

ABSTRACT Mercury has a long history as an antimicrobial agent effective against eukaryotic and prokaryotic organisms. Despite its prolonged use, the basis for mercury toxicity in prokaryotes is not well understood. Archaea, like bacteria, are prokaryotes but they use a simplified version of the eukaryotic transcription apparatus. This study examined the mechanism of mercury toxicity to the archaeal prokaryote Sulfolobus solfataricus. In vivo challenge with mercuric chloride instantaneously blocked cell division, eliciting a cytostatic response at submicromolar concentrations and a cytocidal response at micromolar concentrations. The cytostatic response was accompanied by a 70% reduction in bulk RNA synthesis and elevated rates of degradation of several transcripts, including tfb-1, tfb-2, and lacS. Whole-cell extracts prepared from mercuric chloride-treated cells or from cell extracts treated in vitro failed to support in vitro transcription of 16S rRNAp and lacSp promoters. Extract-mixing experiments with treated and untreated extracts excluded the occurrence of negative-acting factors in the mercury-treated cell extracts. Addition of transcription factor B (TFB), a general transcription factor homolog of eukaryotic TFIIB, to mercury-treated cell extracts restored >50% of in vitro transcription activity. Consistent with this finding, mercuric ion treatment of TFB in vitro inactivated its ability to restore the in vitro transcription activity of TFB-immunodepleted cell extracts. These findings indicate that the toxicity of mercuric ion in S. solfataricus is in part the consequence of transcription inhibition due to TFB-1 inactivation.

Compartmentalization of an all-E. coli Cell-Free Expression System for the Construction of a Minimal Cell

2016 ◽  
Vol 22 (2) ◽  
pp. 185-195 ◽  
Author(s):  
Filippo Caschera ◽  
Vincent Noireaux
Keyword(s):  
Protein Synthesis ◽  
Expression System ◽  
Coli Cell ◽  
Free Expression ◽  
Genetic Circuit ◽  
Translation System ◽  
Minimal Cell ◽  
E Coli ◽  
Synthetic Cell

Cell-free expression is a technology used to synthesize minimal biological cells from natural molecular components. We have developed a versatile and powerful all-E. coli cell-free transcription–translation system energized by a robust metabolism, with the far objective of constructing a synthetic cell capable of self-reproduction. Inorganic phosphate (iP), a byproduct of protein synthesis, is recycled through polysugar catabolism to regenerate ATP (adenosine triphosphate) and thus supports long-lived and highly efficient protein synthesis in vitro. This cell-free TX-TL system is encapsulated into cell-sized unilamellar liposomes to express synthetic DNA programs. In this work, we study the compartmentalization of cell-free TX-TL reactions, one of the aspects of minimal cell module integration. We analyze the signals of various liposome populations by fluorescence microscopy for one and for two reporter genes, and for an inducible genetic circuit. We show that small nutrient molecules and proteins are encapsulated uniformly in the liposomes with small fluctuations. However, cell-free expression displays large fluctuations in signals among the same population, which are due to heterogeneous encapsulation of the DNA template. Consequently, the correlations of gene expression with the compartment dimension are difficult to predict accurately. Larger vesicles can have either low or high protein yields.

scholarly journals Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro.

1993 ◽  
Vol 4 (1) ◽  
pp. 79-92 ◽  
Author(s):  
L Connell-Crowley ◽  
M J Solomon ◽  
N Wei ◽  
J W Harper
Keyword(s):  
Mammalian Cells ◽  
Specific Activity ◽  
Fold Increase ◽  
Cyclin A ◽  
Cyclin B ◽  
Activation Process ◽  
Cyclin Dependent Kinase ◽  
Cell Extracts

p33cdk2 is a serine-threonine protein kinase that associates with cyclins A, D, and E and has been implicated in the control of the G1/S transition in mammalian cells. Recent evidence indicates that cyclin-dependent kinase 2 (Cdk2), like its homolog Cdc2, requires cyclin binding and phosphorylation (of threonine-160) for activation in vivo. However, the extent to which mechanistic details of the activation process are conserved between Cdc2 and Cdk2 is unknown. We have developed bacterial expression and purification systems for Cdk2 and cyclin A that allow mechanistic studies of the activation process to be performed in the absence of cell extracts. Recombinant Cdk2 is essentially inactive as a histone H1 kinase (< 4 x 10(-5) pmol phosphate transferred.min-1 x microgram-1 Cdk2). However, in the presence of equimolar cyclin A, the specific activity is approximately 16 pmol.mon-1 x microgram-1, 4 x 10(5)-fold higher than Cdk2 alone. Mutation of T160 in Cdk2 to either alanine or glutamic acid had little impact on the specific activity of the Cdk2/cyclin A complex: the activity of Cdk2T160E was indistinguishable from Cdk2, whereas that of Cdk2T160A was reduced by five-fold. To determine if the Cdk2/cyclin A complex could be activated further by phosphorylation of T160, complexes were treated with Cdc2 activating kinase (CAK), purified approximately 12,000-fold from Xenopus eggs. This treatment resulted in an 80-fold increase in specific activity. This specific activity is comparable with that of the Cdc2/cyclin B complex after complete activation by CAK (approximately 1600 pmol.mon-1 x microgram-1). Neither Cdk2T160A/cyclin A nor Cdk2T160E/cyclin A complexes were activated further by treatment with CAK. In striking contrast with cyclin A, cyclin B did not directly activate Cdk2. However, both Cdk2/cyclin A and Cdk2/cyclin B complexes display similar activity after activation by CAK. For the Cdk2/cyclin A complex, both cyclin binding and phosphorylation contribute significantly to activation, although the energetic contribution of cyclin A binding is greater than that of T160 phosphorylation by approximately 5 kcal/mol. The potential significance of direct activation of Cdk2 by cyclins with respect to regulation of cell cycle progression is discussed.

Synthesis and characterization of a functional intact IgG in a prokaryotic cell-free expression system

2008 ◽  
Vol 389 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Stephan Frey ◽  
Martin Haslbeck ◽  
Otmar Hainzl ◽  
Johannes Buchner
Keyword(s):  
Mammalian Cells ◽  
Recombinant Expression ◽  
Expression System ◽  
Basic Research ◽  
Medical Diagnostics ◽  
Free Expression ◽  
Translation System ◽  
Functional Antibodies ◽  
The Stability

Abstract Antibodies are an important component of the immune system of higher eukaryotes. Furthermore, they are effective tools in basic research, medical diagnostics and therapy. Recombinant expression of these heterotetrameric, disulfide-bridged proteins is usually performed in mammalian cells. Here, we describe the cell-free expression of a mouse monoclonal antibody, MAK33, in a coupled transcription/translation system, based on an Escherichia coli lysate. Both the heavy and the light chain can be produced efficiently in this setup. However, they fail to form functional antibodies. With a view to overcome folding and oxidation defects, we supplemented the system with the oxidoreductases PDI (protein disulfide isomerase) and DsbC and the ER-specific chaperones Grp94 and BiP; furthermore, we optimized the redox conditions. We found that functional antibodies can only be obtained in the presence of an oxidoreductase. In contrast, the addition of Grp94 and/or BiP had no influence on the productive folding reaction. The comparison of the antibody expressed in vitro with MAK33 expressed in cell culture showed that the in vitro expressed antibody is correctly assembled, disulfide-bridged and shows identical antigen affinity. The stability of the in vitro expressed non-glycosylated IgG is comparable to that of the authentic antibody.

scholarly journals Riboflavin-dependent expression of flavoenzymes of the nicotine regulon of Arthrobacter oxidans

1990 ◽  
Vol 270 (3) ◽  
pp. 673-678 ◽  
Author(s):  
R Brandsch ◽  
V Bichler
Keyword(s):  
Specific Activity ◽  
Enzyme Protein ◽  
Western Blots ◽  
Cell Extracts ◽  
Arthrobacter Oxidans ◽  
Steady State Levels ◽  
Minimal Concentration ◽  
Mutant Cells ◽  
In Cells

In cells of an Arthrobacter oxidans riboflavin-dependent mutant the specific activity of the DL-nicotine-inducible nicregulon enzymes nicotine dehydrogenase (NDH, EC 1.5.99.4), 6-hydroxy-L-nicotine oxidase (6-HLNO, EC 1.5.3.5) and 6-hydroxy-D-nicotine oxidase (6-HDNO, EC 1.5.3.6) was shown to be dependent on the supply of the vitamin in the growth medium. Experiments designed to identify at which level riboflavin directs the biosynthesis of these flavoenzymes revealed that the steady-state levels of enzyme protein analysed on Western blots correlated directly with riboflavin supply from the minimal concentration of 0.5 microns-riboflavin required for growth up to 8 microns-riboflavin. Mutant cells grown at the higher riboflavin concentration showed on dot-blots increased levels of RNA which hybridized to 32P-labelled probes derived from the nic-regulon genes. When cells grown at 2 microns-riboflavin were shifted to 8 microns-riboflavin, 6-HDNO expression increased as indicated by elevated enzyme and RNA levels. When the rates of synthesis of the 6-HDNO and 6-HLNO polypeptides after DL-nicotine induction was analysed in cells grown at 0.5 microns and 8 microns-riboflavin, only cells grown at the higher riboflavin concentration showed on Western blots an accumulation of the polypeptides. No 6-HDNO or 6-HLNO polypeptide was identified in cell extracts from cells grown on 0.5 microns-riboflavin. Pulse-chase experiments with [35S]methionine showed that 6-HDNO- and 6-HLNO synthesis was prevented in cells grown at the low riboflavin concentration. The absence of detectable enzyme levels seemed not to be caused by proteolytic breakdown. Incubation in vitro of apo-6HDNO with low- or high-riboflavin-grown-cell extracts showed no increased proteolytic activity in 0.5 microns-riboflavin-grown cells. From these results it is concluded that riboflavin supply co-regulates the expression of the nicregulon genes at the level of transcription and/or mRNA turnover.

Fast in vitro translation system immobilized on a surface via specific biotinylation of the ribosome

2008 ◽  
Vol 389 (9) ◽  
Author(s):  
Romualdas Stapulionis ◽  
Yuhong Wang ◽  
Graham T. Dempsey ◽  
Rama Khudaravalli ◽  
Karen Margrethe Nielsen ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Single Molecule ◽  
Dynamic Properties ◽  
Kinetic Studies ◽  
Biologically Active ◽  
In Vitro Translation ◽  
23S Rrna ◽  
Translation System ◽  
Peptide Bonds

Abstract The ribosome is the macromolecular machine responsible for translating the genetic code into polypeptide chains. Despite impressive structural and kinetic studies of the translation process, a number of challenges remain with respect to understanding the dynamic properties of the translation apparatus. Single-molecule techniques hold the potential of characterizing the structural and mechanical properties of macromolecules during their functional cycles in real time. These techniques often necessitate the specific coupling of biologically active molecules to a surface. Here, we describe a procedure for such coupling of functionally active ribosomes that permits single-molecule studies of protein synthesis. Oxidation with NaIO4 at the 3′ end of 23S rRNA and subsequent reaction with a biotin hydrazide produces biotinylated 70S ribosomes, which can be immobilized on a streptavidin-coated surface. The surface-attached ribosomes are fully active in poly(U) translation in vitro, synthesizing poly(Phe) at a rate of 3–6 peptide bonds/s per active ribosome at 37°C. Specificity of binding of biotinylated ribosomes to a streptavidin-coated quartz surface was confirmed by observation of individual fluorescently labeled, biotinylated 70S ribosomes, using total internal reflection fluorescence microscopy. Functional interactions of the immobilized ribosomes with various components of the protein synthesis apparatus are shown by use of surface plasmon resonance.

Sign in / Sign up

Export Citation Format

Share Document