9 research outputs found
<i>Pseudomonas</i> exotoxin (PE) productions using the S30-T7 CFPS system originated from two different <i>E</i>. <i>coli</i> strains (BL21 and MRE600).
<p>Reactions were performed with and without the presence of DNA template. (A) Western blot analysis of cell-free reactions demonstrated the production of PE ~ 66 kDa. Purified PE served as positive control (described in Appendix F in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165137#pone.0165137.s001" target="_blank">S1 File</a>.). Arrows indicate the position of PE bands. (B) The therapeutic potency of PE was evaluated on 4T1 cell-line. The viability of the cells was determined by MTT assay. Cell viability values obtained without the presence of purified PE or DNA were set as 100%, and the other values were normalized according to them (error bars represent standard deviation from at least three independent samples).</p
<i>In vitro</i> protein synthesis using cell-free system based on S30-T7 lysate.
<p><i>In vitro</i> protein synthesis using cell-free system based on S30-T7 lysate.</p
A historical overview of improvements made to CFPS procedures over time.
<p>A historical overview of improvements made to CFPS procedures over time.</p
A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis
<div><p>Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from <i>E</i>. <i>coli</i>. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 <i>E</i>. <i>coli</i> strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and <i>E</i>. <i>coli</i> strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.</p></div
Enzyme productions using S30-T7 CFPS systems sourced from two different <i>E</i>. <i>coli</i> strains (BL21 and MRE600).
<p>(A) The produced <i>Renilla</i> luciferase activity was demonstrated by integrating 10 seconds of luminescence measurements (error bars represent standard deviation from at least three independent samples). (B) & (C) TyrBm production was confirmed by monitoring the conversion of 1mM L-Dopa to dopachrome (error bars represent standard deviation from three independent samples). (C) The observed enzymatic activity of TyrBm, produced by the S30-T7 CFPS in a 96-well plate. The three wells to the right present cell-free reaction in the present of DNA template, while in the three wells to the left no DNA template was incorporated into the reaction. The dark color indicates on the conversion of L-Dopa to dopachrome (followed by polymerization and accumulation of melanin), and thus on the production of TyrBm. (D) Temperature effect on cell-free <i>superfolder</i> GFP production efficiency of the S30-T7 CFPS (error bars represent standard deviation from at least four independent samples). The protein production amount was evaluated according to the fluorescence levels. The fluorescence values obtained at 37°C were set to 100%, and all the other values were normalized according to them. Negative controls (N.C.) were reactions without DNA templates. * Significant difference between lysates from the two E. coli strains, where α<0.05 according to a Student's t-Test with a two-tailed distribution with equal variance.</p
A schematic overview of the cell-free protein production process.
<p>A schematic overview of the cell-free protein production process.</p
Remotely Activated Protein-Producing Nanoparticles
The development of responsive nanomaterials, nanoscale
systems that actively respond to stimuli, is one general goal of nanotechnology.
Here we develop nanoparticles that can be controllably triggered to
synthesize proteins. The nanoparticles consist of lipid vesicles filled
with the cellular machinery responsible for transcription and translation,
including amino acids, ribosomes, and DNA caged with a photolabile
protecting group. These particles served as nanofactories capable
of producing proteins including green fluorescent protein (GFP) and
enzymatically
active luciferase. In vitro and in vivo, protein synthesis was spatially
and temporally controllable, and could be initiated by irradiating
micrometer-scale
regions on the time scale of milliseconds. The ability to control
protein synthesis inside nanomaterials may enable new strategies to
facilitate the study of orthogonal proteins in a confined environment
and for remotely activated drug delivery
Proteolytic Nanoparticles Replace a Surgical Blade by Controllably Remodeling the Oral Connective Tissue
Surgical
blades are common medical tools. However, blades cannot
distinguish between healthy and diseased tissue, thereby creating
unnecessary damage, lengthening recovery, and increasing pain. We
propose that surgical procedures can rely on natural tissue remodeling
toolsî—¸enzymes, which are the same tools our body uses to repair
itself. Through a combination of nanotechnology and a controllably
activated proteolytic enzyme, we performed a targeted surgical task
in the oral cavity. More specifically, we engineered nanoparticles
that contain collagenase in a deactivated form. Once placed at the
surgical site, collagenase was released at a therapeutic concentration
and activated by calcium, its biological cofactor that is naturally
present in the tissue. Enhanced periodontal remodeling was recorded
due to enzymatic cleavage of the supracrestal collagen fibers that
connect the teeth to the underlying bone. When positioned in their
new orientation, natural tissue repair mechanisms supported soft and
hard tissue recovery and reduced tooth relapse. Through the combination
of nanotechnology and proteolytic enzymes, localized surgical procedures
can now be less invasive