scholarly journals Cell-free compartmentalized protein synthesis inside double emulsion templated liposomes with in vitro synthesized and assembled ribosomes

2016 ◽  
Vol 52 (31) ◽  
pp. 5467-5469 ◽  
Author(s):  
Filippo Caschera ◽  
Jin Woo Lee ◽  
Kenneth K. Y. Ho ◽  
Allen P. Liu ◽  
Michael C. Jewett
Keyword(s):  
Protein Synthesis ◽  
Double Emulsion ◽  
Free Expression ◽  
Expression Platform ◽  
A Cell

A cell-free expression platform for making bacterial ribosomes encapsulated within giant liposomes was capable of synthesizing sfGFP.

Use of an in Vitro Protein Synthesizing System to Test the Mode of Action of Chloracetamides

Weed Science ◽  
1980 ◽  
Vol 28 (3) ◽  
pp. 334-340 ◽  
Author(s):  
Luanne M. Deal ◽  
J. T. Reeves ◽  
B. A. Larkins ◽  
F. D. Hess
Keyword(s):  
Protein Synthesis ◽  
Sand Culture ◽  
Leucine Incorporation ◽  
Insoluble Protein ◽  
In Vitro System ◽  
A Cell ◽  
Protein Incorporation ◽  
Vitro Protein

The effects of chloracetamides on protein synthesis were studied both in vivo and in vitro. Four chloracetamide herbicides, alachlor [2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide], metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide], CDAA (N–N-diallyl-2-chloroacetamide), and propachlor (2-chloro-N-isopropylacetanilide) were tested for inhibition of [3H]-leucine incorporation into protein. Incorporation of3H-leucine into trichloroacetic acid (TCA)-insoluble protein was inhibited in oat (Avena sativaL. ‘Victory’) seedlings grown in sand culture and treated 12 h at 1 × 10−4M with these chloracetamides. The herbicides were also tested in a cell-free protein synthesizing system containing polyribosomes purified from oat root cytoplasm. These herbicides had no effect on the rates of polypeptide elongation nor on the synthesis of specific polypeptides when herbicides (1 × 10−4M) were added directly to the system. Polypeptide formation was inhibited 89% when 1 × 10−4M cycloheximide was added during translation. Cytoplasmic polyribosomes were isolated from oat roots treated 12 h with 1 × 10−4M herbicide. Translation rates and products were not altered when these polyribosomes were added to the in vitro system. Protein synthesis is inhibited when tested in an in vivo system; however, the inhibition does not occur during the translation of mRNA into protein.

scholarly journals Efficacy of a Potential Trivalent Vaccine Based on Hc Fragments of Botulinum Toxins A, B, and E Produced in a Cell-Free Expression System

2010 ◽  
Vol 17 (5) ◽  
pp. 784-792 ◽  
Author(s):  
R. Zichel ◽  
A. Mimran ◽  
A. Keren ◽  
A. Barnea ◽  
I. Steinberger-Levy ◽  
...  
Keyword(s):  
Expression System ◽  
Free Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Dose ◽  
Botulinum Toxins ◽  
Free System ◽  
Toxin Complex ◽  
A Cell ◽  
Cell Free Expression System

ABSTRACT Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni+ affinity chromatography. Mice immunized with three injections containing 5 μg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 105 against the native toxin complex, which enabled protection against a high-dose toxin challenge (103 to 106 mouse 50% lethal dose [MsLD50]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 105 MsLD50 toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

scholarly journals Directed Evolution of Proteins throughIn VitroProtein Synthesis in Liposomes

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Takehiro Nishikawa ◽  
Takeshi Sunami ◽  
Tomoaki Matsuura ◽  
Tetsuya Yomo
Keyword(s):  
Protein Synthesis ◽  
Directed Evolution ◽  
Single Copy ◽  
Functional Evaluation ◽  
Future Directions ◽  
Pure System ◽  
Protein Functions ◽  
A Cell ◽  
Evolution Of Proteins

Directed evolution of proteins is a technique used to modify protein functions through “Darwinian selection.”In vitrocompartmentalization (IVC) is anin vitrogene screening system for directed evolution of proteins. IVC establishes the link between genetic information (genotype) and the protein translated from the information (phenotype), which is essential for all directed evolution methods, by encapsulating both in a nonliving microcompartment. Herein, we introduce a new liposome-based IVC system consisting of a liposome, the protein synthesis using recombinant elements (PURE) system and a fluorescence-activated cell sorter (FACS) used as a microcompartment,in vitroprotein synthesis system, and high-throughput screen, respectively. Liposome-based IVC is characterized byin vitroprotein synthesis from a single copy of a gene in a cell-sized unilamellar liposome and quantitative functional evaluation of the synthesized proteins. Examples of liposome-based IVC for screening proteins such as GFP andβ-glucuronidase are described. We discuss the future directions for this method and its applications.

Effect of trna from 5-azacytidine-treated hamster fibrosarcoma cells on protein synthesis in vitro in a cell-free system

1976 ◽  
Vol 25 (4) ◽  
pp. 389-392 ◽  
Author(s):  
Richard L. Momparler ◽  
Stuart Siegel ◽  
Felicidad Avila ◽  
Thomas Lee ◽  
Myron Karon
Keyword(s):  
Protein Synthesis ◽  
Free System ◽  
Cell Free System ◽  
Fibrosarcoma Cells ◽  
A Cell ◽  
Hamster Fibrosarcoma

scholarly journals Cloning-Independent Expression and Analysis of ω-Transaminases by Use of a Cell-Free Protein Synthesis System

2010 ◽  
Vol 76 (18) ◽  
pp. 6295-6298 ◽  
Author(s):  
Yong-Chan Kwon ◽  
Kyung-Ho Lee ◽  
Ho-Cheol Kim ◽  
Kyuboem Han ◽  
Joo-Hyun Seo ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Function ◽  
Free Expression ◽  
Synthesis System ◽  
Free Protein ◽  
Enzyme Substrate ◽  
Information Gap ◽  
A Cell ◽  
Protein Synthesis System ◽  
Cell Free Protein Synthesis

ABSTRACT Herewith we report the expression and screening of microbial enzymes without involving cloning procedures. Computationally predicted putative ω-transaminase (ω-TA) genes were PCR amplified from the bacterial colonies and expressed in a cell-free protein synthesis system for subsequent analysis of their enzymatic activity and substrate specificity. Through the cell-free expression analysis of the putative ω-TA genes, a number of enzyme-substrate pairs were identified in a matter of hours. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function to accelerate the discovery of novel enzymes.

Glycoproteins on the surface of smooth muscle cells involved in their interaction with type V collagen

1985 ◽  
Vol 63 (11) ◽  
pp. 1176-1182 ◽  
Author(s):  
James R. A. Leushner ◽  
M. Daria Haust
Keyword(s):  
Protein Synthesis ◽  
Cyanogen Bromide ◽  
Attachment Site ◽  
Membrane Glycoproteins ◽  
Type V Collagen ◽  
Attachment Region ◽  
A Cell ◽  
Type V ◽  
In Vitro Interaction

Type V collagen is a major component of the pericellular coat of smooth cells (SMC). The purpose of the present study was to assess biochemically the nature of an in vitro interaction between bovine aortic SMC and type V collagen from the same source. This interaction was originally shown to be mediated by a cell-surface glycoconjugate. Data obtained in the present study suggests that the binding system consists of integral membrane glycoproteins which act alone or in combination with a surface glycolipid in type V attachment. The nature of this system was indicated by the finding of 80 000 and 50 000 components in the plasma membrane fractions which were specifically retained by type V collagen – Sepharose columns and incorporated both methionine and mannose label. Moreover, inhibition of protein synthesis lowered SMC attachment by 25%. The mannose label associated with these components was probably in the form of a simple oligosaccharide at the attachment site since it bound to concanavalinA (ConA) and was sensitive to endoglycosidase H. Iodinated ConA labelling indicated elevated levels of these components were associated with SMC – type V collagen interaction. The attachment region on the type V molecule was localized within the cyanogen bromide peptide 6 of the α2 (V) chain.

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.

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