Synthetic cells synthesize therapeutic proteins inside tumors

The existing dogma is that protein medicines need to be produced in large factories, and then injected to the patient. We propose that miniature artificial inert factories can be injected to the patient, to produce a protein of interest directly in the diseased tissue. We engineered artificial cell-like particles with an autonomous capacity to synthesize protein drugs after receiving an external signal. The protein is tuned to the patient\u27s needs based on a predetermined DNA code we incorporate inside the particles. This approach increases treatment efficiency and reduces adverse effects to healthy tissues. We developed a new T7-S30 based cell-free protein synthesis system, which contains all the transcription and translation machines and molecules required for protein production (Krinsky et al., PloS one 2016). This system was used to prepare liposomes that act as artificial cells, capable of producing proteins autonomously in response to a physical trigger. Functional enzymes (luciferase and tyrosinase) and fluorescent proteins (GFP) were successfully produced using the new cell-free protein synthesis system and inside the particles both in vitro and in vivo. In addition, we demonstrated the therapeutic capabilities of the protein producing particles by producing Pseudomonas exotoxin A, an extremely potent protein, for treating cancer. Applying the particles on 4T1 cells (a triple-negative breast cancer cell-line) in vitro or injecting them into a 4T1-induced tumor in vivo, resulted in high cytotoxicity due to the effective production of the therapeutic protein inside the vesicles (Krinsky et al. Advanced Healthcare Materials, 2017). Synthetic cells serve as autonomous, trigger-able, artificial particles that produces a variety of proteins. This platform has promise to address a wide range of fundamental questions associated with protein synthesis in nature, as well as applicative protein delivery needs. Please click Additional Files below to see the full abstract

Protein Engineering of Toluene Monooxygenases for Synthesis of Chiral Sulfoxides▿

Enantiopure sulfoxides are valuable asymmetric starting materials and are important chiral auxiliaries in organic synthesis. Toluene monooxygenases (TMOs) have been shown previously to catalyze regioselective hydroxylation of substituted benzenes and phenols. Here we show that TMOs are also capable of performing enantioselective oxidation reactions of aromatic sulfides. Mutagenesis of position V106 in the α-hydroxylase subunit of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 and the analogous position I100 in toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 improved both rate and enantioselectivity. Variant TomA3 V106M of TOM oxidized methyl phenyl sulfide to the corresponding sulfoxide at a rate of 3.0 nmol/min/mg protein compared with 1.6 for the wild-type enzyme, and the enantiomeric excess (pro-S) increased from 51% for the wild type to 88% for this mutant. Similarly, T4MO variant TmoA I100G increased the wild-type oxidation rate by 1.7-fold, and the enantiomeric excess rose from 86% to 98% (pro-S). Both wild-type enzymes showed lower activity with methyl para-tolyl sulfide as a substrate, but the improvement in the activity and enantioselectivity of the mutants was more dramatic. For example, T4MO variant TmoA I100G oxidized methyl para-tolyl sulfide 11 times faster than the wild type did and changed the selectivity from 41% pro-R to 77% pro-S. A correlation between regioselectivity and enantioselectivity was shown for TMOs studied in this work. Using in silico homology modeling, it is shown that residue I100 in T4MO aids in steering the substrate into the active site at the end of the long entrance channel. It is further hypothesized that the main function of V106 in TOM is the proper positioning or docking of the substrate with respect to the diiron atoms. The results from this work suggest that when the substrate is not aligned correctly in the active site, the oxidation rate is decreased and enantioselectivity is impaired, resulting in products with both chiral configurations

Rapid Methods for High-Throughput Detection of Sulfoxides▿

Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene ortho-monooxygenase in a saturation mutagenesis library, using methyl-p-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment

Preparation of S30-T7 lysate.

<p>Preparation of S30-T7 lysate.</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

<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

A historical overview of improvements made to CFPS procedures over time.

<p>A historical overview of improvements made to CFPS procedures over time.</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