In vitro translation in a hybrid cell free lysate with exogenous cellular ribosomes

2015 ◽  
Vol 467 (3) ◽  
pp. 387-398 ◽  
Author(s):  
Baptiste Panthu ◽  
Didier Décimo ◽  
Laurent Balvay ◽  
Théophile Ohlmann
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Hybrid System ◽  
Hybrid Cell ◽  
Cell Types ◽  
In Vitro Translation ◽  
Translation System ◽  
Different Origins ◽  
Cell Free Protein Synthesis

Cell free protein synthesis systems (CFPS) have been widely used to express proteins and to explore the pathways of gene expression. In the present manuscript, we describe the design of a novel adaptable hybrid in vitro translation system which is assembled with ribosomes isolated from many different origins. We first show that this hybrid system exhibits all important features such as efficiency, sensitivity, reproducibility and the ability to translate specialized mRNAs in less than 1 h. In addition, the unique design of this cell free assay makes it highly adaptable to utilize ribosomes isolated from many different organs, tissues or cell types.

scholarly journals Identification of Mimotope Peptides Which Bind to the Mycotoxin Deoxynivalenol-Specific Monoclonal Antibody

1999 ◽  
Vol 65 (8) ◽  
pp. 3279-3286 ◽  
Author(s):  
Qiaoping Yuan ◽  
James J. Pestka ◽  
Brandon M. Hespenheide ◽  
Leslie A. Kuhn ◽  
John E. Linz ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Synthetic Peptide ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Translation System

ABSTRACT Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentous phages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5 specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically to mAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbit reticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON.

Changes in Ribosome Organization and Messenger RNA Abundance in Ripening Tomato Fruits

1984 ◽  
Vol 11 (3) ◽  
pp. 225 ◽  
Author(s):  
J Spiers ◽  
CJ Brady ◽  
D Grierson ◽  
E Lee
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Rna ◽  
Messenger Rna ◽  
In Vitro Translation ◽  
Translation System ◽  
Active Protein ◽  
Tomato Fruits ◽  
Rna And Protein Synthesis ◽  
Early Ripening

The involvement of RNA and protein synthesis in fruit ripening was investigated. Mature-green tomato fruits were found to contain about 30% of their ribosomal RNA in polyribosomes. At the 'breaker' or early ripening stage, about 50% of rRNA was in polyribosomes and this distribution of rRNA was maintained until fruits were fully ripe. The continued presence of polyribosomes is consistent with active protein synthesis persisting through and beyond the climacteric period when the wall-hydrolysing enzyme polygalacturonase accumulates and fruits soften, synthesize lycopene and undergo other ripening related changes. Poly(A)-containing RNA purified from polyribosomes extracted from individual fruits was used to prime the synthesis of [35S]methionine-labelled polypeptides by a wheat germ in vitro translation system. The pattern of polypeptides synthesized in response to RNA from mature-green fruits differed from that given by RNA from ripening fruits. The majority of changes were found to occur within approximately 48 h of the increase of ethylene synthesis and were apparent in all fruits with any pink or red colour. Similar results were obtained by translating total cellular RNA and total polyribosomal RNA indicating that the major RNA species shown in this study to change in abundance during ripening are polyadenylated, and hence most probably cytoplasmic, and do not accumulate as 'stored messages' outside of the polyribosomes. The differences between green and ripening fruits in polyribosome profiles were demonstrated in two cultivars of tomato. Differences in mRNA populations between green and ripe fruits were found in three cultivars.

scholarly journals Synthetic Biology Bicistronic Designs Support Gene Expression Equally Well in vitro and in vivo

2020 ◽  
Vol 17 (1) ◽  
pp. 13-20
Author(s):  
Owen Koucky ◽  
Jacob Wagner ◽  
Sofia Aguilera ◽  
Benjamin Bashaw ◽  
Queena Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Synthetic Biology ◽  
Protein Production ◽  
Persistent Problem ◽  
Free Protein ◽  
Microfluidic Droplets ◽  
Cell Free Protein Synthesis

Synthetic biology integrates molecular biology tools and an engineering mindset to address challenges in medicine, agriculture, bioremediation, and biomanufacturing. A persistent problem in synthetic biology has been designing genetic circuits that produce predictable levels of protein. In 2013, Mutalik and colleagues developed bicistronic designs (BCDs) that make protein production more predicable in bacterial cells (in vivo). With the growing interest in producing proteins outside of cells (in vitro), we wanted to know if BCDs would work as predictably in cell-free protein synthesis (CFPS) as they do in E. coli cells. We tested 20 BCDs in CFPS and found they performed very similarly in vitro and in vivo. As a step toward developing methods for protein production in artificial cells, we also tested 3 BCDs inside nanoliter-scaled microfluidic droplets. The BCDs worked well in the microfluidic droplets, but their relative protein production levels were not as predictable as expected. These results suggest that the conditions under which gene expression happens in droplets result in a different relationship between genetic control elements such as BCDs and protein production than exists in batch CFPS or in cells. KEYWORDS: Bicistronic Design; Synthetic Biology; Cell-Free Protein Synthesis; Microfluidics

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.

Enhancement of in-vitro Translation of Eukaryotic RNAs by Cyclic AMP

1983 ◽  
Vol 38 (3-4) ◽  
pp. 277-281
Author(s):  
M. E. John ◽  
W. Knöchel
Keyword(s):  
Protein Synthesis ◽  
Secondary Structure ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Methyl Mercury ◽  
In Vitro Translation ◽  
Translation System ◽  
Reticulocyte Lysate ◽  
Stimulation Of

Addition of cyclic AMP to normal rabbit reticulocyte lysate brings about substantial increase in protein synthesis programmed by both nuclear and polysomal eukaryotic RNAs. The increase in synthesis is largely nonspecific and the in-vitro translation system is rendered viable for longer periods of time than in the absence of cyclic AMP. This stimulation of translation could be due to the inhibition of protein kinases that are initially activated by secondary structure of RNAs. However, the ability of cyclic AMP to further stimulate methyl mercury or heat denatured RNA indicates that additional mechanisms may be involved in the enhancement of translation

scholarly journals Ectopic Overexpression of Wild-Type and Mutant hipA Genes in Escherichia coli: Effects on Macromolecular Synthesis and Persister Formation

2006 ◽  
Vol 188 (11) ◽  
pp. 3826-3836 ◽  
Author(s):  
Shaleen B. Korch ◽  
Thomas M. Hill
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Prolonged Exposure ◽  
Physiological Role ◽  
In Vitro Translation ◽  
Translation System ◽  
Wild Type ◽  
Macromolecular Synthesis ◽  
Dormant State

ABSTRACT Persistence is an epigenetic trait that allows a small fraction of bacteria, approximately one in a million, to survive prolonged exposure to antibiotics. In Escherichia coli an increased frequency of persisters, called “high persistence,” is conferred by mutations in the hipA gene, which encodes the toxin entity of the toxin-antitoxin module hipBA. The high-persistence allele hipA7 was originally identified because of its ability to confer high persistence, but little is known about the physiological role of the wild-type hipA gene. We report here that the expression of wild-type hipA in excess of hipB inhibits protein, RNA, and DNA synthesis in vivo. However, unlike the RelE and MazF toxins, HipA had no effect on protein synthesis in an in vitro translation system. Moreover, the expression of wild-type hipA conferred a transient dormant state (persistence) to a sizable fraction of cells, whereas the rest of the cells remained in a prolonged dormant state that, under appropriate conditions, could be fully reversed by expression of the cognate antitoxin gene hipB. In contrast, expression of the mutant hipA7 gene in excess of hipB did not markedly inhibit protein synthesis as did wild-type hipA and yet still conferred persistence to ca. 10% of cells. We propose that wild-type HipA, upon release from HipB, is able to inhibit macromolecular synthesis and induces a bacteriostatic state that can be reversed by expression of the hipB gene. However, the ability of the wild-type hipA gene to generate a high frequency of persisters, equal to that conferred by the hipA7 allele, may be distinct from the ability to block macromolecular synthesis.

scholarly journals Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Liyuan Zhang ◽  
Xiaomei Lin ◽  
Ting Wang ◽  
Wei Guo ◽  
Yuan Lu
Keyword(s):  
Protein Synthesis ◽  
Protein Production ◽  
Influential Factors ◽  
Competent Cell ◽  
Free Protein ◽  
Application Range ◽  
E Coli ◽  
Short Time ◽  
Cell Free Protein Synthesis

AbstractCell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

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