The Experts below are selected from a list of 173172 Experts worldwide ranked by ideXlab platform

Michael C. Jewett - One of the best experts on this subject based on the ideXlab platform.

  • cell free Protein Synthesis enables one pot cascade biotransformation in an aqueous organic biphasic system
    Biotechnology and Bioengineering, 2020
    Co-Authors: Wanqiu Liu, Michael C. Jewett
    Abstract:

    Biocatalytic cascade reactions have become increasingly important and useful for chemical Synthesis. However, biocatalysts are often incompatible with organic solvents, which prohibits many cascade reactions involving nonpolar substrates. In this study, we used Cell-Free Protein Synthesis (CFPS) to express enzymes in an aqueous-organic biphasic system for the construction of an artificial enzymatic pathway. CFPS-expressed enzymes without purification performed efficiently to convert styrene (below 20 mM) to (S)-1-phenyl-1,2-ethanediol (two steps in one pot) with 100% conversion. In addition, our CFPS system showed great tolerance to different organic solvents, and, importantly, the entire biocatalytic system can be consistently scaled up without a reduction of the substrate conversion rate. We, therefore, anticipate that our Cell-Free approach will make a possible cost-effective, high-yielding Synthesis of valuable chemicals.

  • escherichia coli based cell free Protein Synthesis protocols for a robust flexible and accessible platform technology
    Journal of Visualized Experiments, 2019
    Co-Authors: Max Z Levine, Michael C. Jewett, Nicole E Gregorio, Katharine R Watts, Javin P Oza
    Abstract:

    Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging Proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for Protein expression capable of producing 900 µg/mL or more of super folder green fluorescent Protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per µg of Protein produced or $0.019 per µL of reaction. Additionally, the Protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the Protein expression outcomes desired.

  • development of a pseudomonas putida cell free Protein Synthesis platform for rapid screening of gene regulatory elements
    Synthetic Biology, 2018
    Co-Authors: He Wang, Michael C. Jewett
    Abstract:

    Cell-Free Protein Synthesis (CFPS) systems enable the production of Protein without the use of living, intact cells. An emerging area of interest is to use CFPS systems to characterize individual elements for genetic programs [e.g. promoters, ribosome binding sites (RBS)]. To enable this research area, robust CFPS systems must be developed from new chassis organisms. One such chassis is the Gram-negative Pseudomonas bacteria, which have been studied extensively for their diverse metabolism with promises in the field of bioremediation and bioSynthesis. Here, we report the development and optimization of a high-yielding (198 ± 5.9 µg/ml) batch CFPS system from Pseudomonas putida ATCC 12633. Importantly, both circular and linear DNA templates can be applied directly to the CFPS reaction to program Protein Synthesis. Therefore, it is possible to prepare hundreds or even thousands of DNA templates without time-consuming cloning work. This opens the possibility to rapidly assess and validate genetic part performance in vitro before performing experiments in cells. To validate the P. putida CFPS system as a platform for prototyping genetic parts, we designed and constructed a library consisting of 15 different RBSs upstream of the reporter Protein sfGFP, which covered an order of magnitude range in expression. Looking forward, our P. putida CFPS platform will not only expand the Protein Synthesis toolkit for synthetic biology but also serve as a platform in expediting the screening and prototyping of gene regulatory elements.

  • establishing a high yielding streptomyces based cell free Protein Synthesis system
    Biotechnology and Bioengineering, 2017
    Co-Authors: He Wang, Yongchan Kwon, Michael C. Jewett
    Abstract:

    Cell-Free Protein Synthesis (CFPS) has emerged as a powerful platform for applied biotechnology and synthetic biology, with a range of applications in synthesizing Proteins, evolving Proteins, and prototyping genetic circuits. To expand the current CFPS repertoire, we report here the development and optimization of a Streptomyces-based CFPS system for the expression of GC-rich genes. By developing a streamlined crude extract preparation protocol and optimizing reaction conditions, we were able to achieve active enhanced green fluorescent Protein (EGFP) yields of greater than 50 μg/mL with batch reactions lasting up to 3 h. By adopting a semi-continuous reaction format, the EGFP yield could be increased to 282 ± 8 μg/mL and the reaction time was extended to 48 h. Notably, our extract preparation procedures were robust to multiple Streptomyces lividans and Streptomyces coelicolor strains, although expression yields varied. We show that our optimized Streptomyces lividans system provides benefits when compared to an Escherichia coli-based CFPS system for increasing percent soluble Protein expression for four Streptomyces-originated high GC-content genes that are involved in bioSynthesis of the nonribosomal peptides tambromycin and valinomycin. Looking forward, we believe that our Streptomyces-based CFPS system will contribute significantly towards efforts to express complex natural product gene clusters (e.g., nonribosomal peptides and polyketides), providing a new avenue for obtaining and studying natural product bioSynthesis pathways. Biotechnol. Bioeng. 2017;114: 1343-1353. © 2017 Wiley Periodicals, Inc.

  • in vitro reconstruction of nonribosomal peptide bioSynthesis directly from dna using cell free Protein Synthesis
    ACS Synthetic Biology, 2017
    Co-Authors: Anthony Whitney Goering, Michael C. Jewett, Jian Li, Ryan A Mcclure, Regan J Thomson, Neil L. Kelleher
    Abstract:

    Genome sequencing has revealed that a far greater number of natural product biosynthetic pathways exist than there are known natural products. To access these molecules directly and deterministically, a new generation of heterologous expression methods is needed. Cell-Free Protein Synthesis has not previously been used to study nonribosomal peptide bioSynthesis, and provides a tunable platform with advantages over conventional methods for Protein expression. Here, we demonstrate the use of Cell-Free Protein Synthesis to biosynthesize a cyclic dipeptide with correct absolute stereochemistry. From a single-pot reaction, we measured the expression of two nonribosomal peptide synthetases larger than 100 kDa, and detected high-level production of a diketopiperazine. Using quantitative LC–MS and synthetically prepared standard, we observed production of this metabolite at levels higher than previously reported from cell-based recombinant expression, approximately 12 mg/L. Overall, this work represents a first s...

James R. Swartz - One of the best experts on this subject based on the ideXlab platform.

  • toward a genome scale sequence specific dynamic model of cell free Protein Synthesis in escherichia coli
    Metabolic Engineering Communications, 2020
    Co-Authors: Nicholas Horvath, James R. Swartz, Kara Calhoun, Michael Vilkhovoy, Joseph A Wayman, Jeffrey D Varner
    Abstract:

    Abstract In this study, we developed a dynamic mathematical model of E. coli Cell-Free Protein Synthesis (CFPS). Model parameters were estimated from a dataset consisting of glucose, organic acids, energy species, amino acids, and Protein product, chloramphenicol acetyltransferase (CAT) measurements. The model was successfully trained to simulate these measurements, especially those of the central carbon metabolism. We then used the trained model to evaluate the performance, e.g., the yield and rates of Protein production. CAT was produced with an energy efficiency of 12%, suggesting that the process could be further optimized. Reaction group knockouts showed that Protein productivity was most sensitive to the oxidative phosphorylation and glycolysis/gluconeogenesis pathways. Amino acid bioSynthesis was also important for productivity, while overflow metabolism and TCA cycle affected the overall system state. In addition, translation was more important to productivity than transcription. Finally, CAT production was robust to allosteric control, as were most of the predicted metabolite concentrations; the exceptions to this were the concentrations of succinate and malate, and to a lesser extent pyruvate and acetate, which varied from the measured values when allosteric control was removed. This study is the first to use kinetic modeling to predict dynamic Protein production in a Cell-Free E. coli system, and could provide a foundation for genome scale, dynamic modeling of Cell-Free E. coli Protein Synthesis.

  • sequence specific modeling of e coli cell free Protein Synthesis
    ACS Synthetic Biology, 2018
    Co-Authors: Michael Vilkhovoy, James R. Swartz, Kara Calhoun, Nicholas Horvath, Chehsiao Shih, Joseph A Wayman, Jeffrey D Varner
    Abstract:

    Cell-Free Protein Synthesis (CFPS) is a widely used research tool in systems and synthetic biology. However, if CFPS is to become a mainstream technology for applications such as point of care manufacturing, we must understand the performance limits and costs of these systems. Toward this question, we used sequence specific constraint based modeling to evaluate the performance of E. coli Cell-Free Protein Synthesis. A core E. coli metabolic network, describing glycolysis, the pentose phosphate pathway, energy metabolism, amino acid bioSynthesis, and degradation was augmented with sequence specific descriptions of transcription and translation and effective models of promoter function. Model parameters were largely taken from literature; thus the constraint based approach coupled the transcription and translation of the Protein product, and the regulation of gene expression, with the availability of metabolic resources using only a limited number of adjustable model parameters. We tested this approach by s...

  • toward a genome scale sequence specific dynamic model of cell free Protein Synthesis in escherichia coli
    bioRxiv, 2017
    Co-Authors: Nicholas Horvath, James R. Swartz, Kara Calhoun, Michael Vilkhovoy, Joseph A Wayman, Jeffrey D Varner
    Abstract:

    Cell-Free Protein expression systems have become widely used in systems and synthetic biology. In this study, we developed an ensemble of dynamic E. coli Cell-Free Protein Synthesis (CFPS) models. Model parameters were estimated from a training dataset for the Cell-Free production of a Protein product, chloramphenicol acetyltransferase (CAT). The dataset consisted of measurements of glucose, organic acids, energy species, amino acids, and CAT. The ensemble accurately predicted these measurements, especially those of the central carbon metabolism. We then used the trained model to evaluate the optimality of Protein production. CAT was produced with an energy efficiency of 12%, suggesting that the process could be further optimized. Reaction group knockouts showed that Protein productivity and the metabolism as a whole depend most on oxidative phosphorylation and glycolysis and gluconeogenesis. Amino acid bioSynthesis is also important for productivity, while the overflow metabolism and TCA cycle affect the overall system state. In addition, the translation rate is shown to be more important to productivity than the transcription rate. Finally, CAT production was robust to allosteric control, as was most of the network, with the exception of the organic acids in central carbon metabolism. This study is the first to use kinetic modeling to predict dynamic Protein production in a Cell-Free E. coli system, and should provide a foundation for genome scale, dynamic modeling of Cell-Free E. coli Protein Synthesis.

  • sequence specific modeling of e coli cell free Protein Synthesis
    bioRxiv, 2017
    Co-Authors: Michael Vilkhovoy, James R. Swartz, Kara Calhoun, Nicholas Horvath, Chehsiao Shih, Joseph A Wayman, Jeffrey D Varner
    Abstract:

    Cell-Free Protein Synthesis (CFPS) has become a widely used research tool in sys- tems and synthetic biology. In this study, we used sequence specific constraint based modeling to evaluate the performance of an E. coli Cell-Free Protein Synthesis system. A core E. coli metabolic model, describing glycolysis, the pentose phosphate pathway, energy metabolism, amino acid bioSynthesis and degradation was augmented with sequence specific descriptions of transcription and translation with effective models of promoter function. Thus, sequence specific constraint based modeling explicitly couples transcription and translation processes and the regulation of gene expression with the availability of metabolic resources. We tested this approach by simulating the expression of two model Proteins: chloramphenicol acetyltransferase and dual emission green fluorescent Protein, for which we have training data sets; we then expanded the simulations to a range of therapeutically relevant Proteins. Protein expression simulations were consistent with measurements for a variety of cases. We then compared optimal and experimentally constrained CFPS reactions, which sug- gested the experimental system over-consumed glucose and had suboptimal oxidative phosphorylation activity. Lastly, global sensitivity analysis identified the key metabolic processes that controlled the CFPS productivity, energy efficiency, and carbon yield. In summary, sequence specific constraint based modeling of CFPS offered a novel means to a priori estimate the performance of a Cell-Free system, using only a limited number of of adjustable parameters. In this study we modeled the production of a single Protein, however this approach could be extended to multi-Protein synthetic circuits, RNA circuits or small molecule production.

  • using e coli based cell free Protein Synthesis to evaluate the kinetic performance of an orthogonal trna and aminoacyl trna synthetase pair
    Biochemical and Biophysical Research Communications, 2013
    Co-Authors: Cem Albayrak, James R. Swartz
    Abstract:

    Abstract Even though the orthogonal tRNA and aminoacyl-tRNA synthetase pairs derived from the archaeon Methanocaldococcus jannaschii have been used for many years for site-specific incorporation of non-natural amino acids (nnAAs) in Escherichia coli, their kinetic parameters have not been evaluated. Here we use a Cell-Free Protein Synthesis (CFPS) system to control the concentrations of the orthogonal components in order to evaluate their performance while supporting Synthesis of modified Proteins (i.e. Proteins with nnAAs). Titration experiments and estimates of turnover numbers suggest that the orthogonal synthetase is a very slow catalyst when compared to the native E. coli synthetases. The estimated kcat for the orthogonal synthetase specific to the nnAA p-propargyloxyphenylalanine (pPaF) is 5.4 × 10−5 s−1. Thus, this catalyst may be the limiting factor for nnAA incorporation when using this approach. These titration experiments also resulted in the highest reported Cell-Free accumulation of two different modified Proteins (450 ± 20 μg/ml CAT109pAzF and 428 ± 2 μg/ml sfGFP23pPaF) using the standard KC6 cell extract and either the PANOx SP or the inexpensive Glu NMP Cell-Free recipe.

Dong Myung Kim - One of the best experts on this subject based on the ideXlab platform.

  • Comparative evaluation of two Cell-Free Protein Synthesis systems derived from Escherichia coli for genetic code reprogramming
    Journal of biotechnology, 2012
    Co-Authors: Ki Baek Lee, Dong Myung Kim, Ho-cheol Kim, Taek Jin Kang, Hiroaki Suga
    Abstract:

    Genetic codes can be reprogrammed to code for non-Proteinogenic amino acids during Protein Synthesis. Technologically, these non-Proteinogenic amino acids are incorporated into Proteins by artificially charging them to suppressor-tRNAs that can reprogram the existing codons. Several methods and systems for genetic code reprogramming have been reported including methods for charging non-Proteinogenic amino acids to tRNA molecules, codons for reprogramming, and systems for Protein Synthesis. However, there has been no systematic, comparative evaluation of Cell-Free Protein Synthesis systems in genetic code reprogramming for their efficiencies and robustness even with their potential usefulness in the field. Here we compare two Cell-Free Protein Synthesis systems, the crude S12 and PURE system, with the codon systems, non-Proteinogenic amino acids, and the positions in the Protein for reprogramming as variables. We show that the combined use of CCCG four-nucleotide codon that is newly developed in this study and the crude S12 system is the most reliable and robust method of choice, while the use of traditional UAG amber stop codon along with an RNA aptamer toward peptide release factor 1 can yield the most plentiful product with certain variations.

  • a highly efficient and economical cell free Protein Synthesis system using the s12 extract of escherichia coli
    Biotechnology and Bioprocess Engineering, 2008
    Co-Authors: Taewan Kim, Ho-cheol Kim, Dong Myung Kim
    Abstract:

    We have developed an economical and simple Cell-Free Protein Synthesis system that produces milligram quantities of Proteins in a milliliter batch reaction. In this system, the S12 extract, which was prepared from glucose-adapted cells, was employed and glucose alone was successfully used for the efficient and stable regeneration of ATP. The ATP level in the reaction mixture remained stable over a remarkably extended reaction period, which enabled prolonged Protein Synthesis, and the issues associated with proton accumulation and amino acid depletion were simultaneously addressed. Under the reaction conditions established in this study, Protein Synthesis continued for 6 h and the amount of the accumulated Protein reached 1.8 mg/mL.

  • prolonged cell free Protein Synthesis using dual energy sources combined use of creatine phosphate and glucose for the efficient supply of atp and retarded accumulation of phosphate
    Biotechnology and Bioengineering, 2007
    Co-Authors: Taewan Kim, Yongchan Kwon, Cha Yong Choi, Jungwon Keum, Juyoung Byun, Kyungho Lee, Dong Myung Kim
    Abstract:

    The accumulation of inorganic phosphate inhibits Protein Synthesis in Cell-Free Protein Synthesis reactions that are energized by high-energy-phosphate-containing compounds. This study developed a new scheme for supplying energy using dual energy sources to enhance the regeneration of ATP and lower the rate of phosphate accumulation. In the proposed scheme, where creatine phosphate (CP) and glucose were simultaneously used as the energy sources, the phosphate released from the CP was subsequently used in the glycolytic pathway for the utilization of the glucose, which enhanced the ATP supply and reduced the rate of inorganic phosphate accumulation. When tested against different Proteins, the developed method produced 2–3 times more Protein than the conventional ATP regeneration methods using single energy sources. Biotechnol. Bioeng. 2007; 97: 1510–1515. © 2007 Wiley Periodicals, Inc.

  • an economical and highly productive cell free Protein Synthesis system utilizing fructose 1 6 bisphosphate as an energy source
    Journal of Biotechnology, 2007
    Co-Authors: Taewan Kim, Ho-cheol Kim, Cha Yong Choi, Jungwon Keum, Dong Myung Kim
    Abstract:

    In this study, we describe the development of a cost effective and highly productive Cell-Free Protein Synthesis system derived from Escherichia coli. Through the use of an optimal energy source and cell extract, approximately 1.3 mg/mL of Protein was generated from a single batch reaction at greatly reduced reagent costs. Compared to previously reported systems, the described method yields approximately 14-fold higher productivity per unit reagent cost making this Cell-Free Synthesis technique a promising alternative for more efficient Protein production.

  • simple procedures for the construction of a robust and cost effective cell free Protein Synthesis system
    Journal of Biotechnology, 2006
    Co-Authors: Taewan Kim, Cha Yong Choi, Jungwon Keum, Changgil Park, Dong Myung Kim
    Abstract:

    In this study, as a part of our efforts to improve the robustness and economical feasibility of Cell-Free Protein Synthesis, we developed a simple method of preparing the cell extracts used for catalyzing Cell-Free Protein Synthesis reactions. We found that the high-speed centrifugation, pre-incubation, and dialysis steps of the conventional procedures could be omitted without losing the translational activity of the resulting cell extract. Instead, a simple centrifugation step at low speed (12,000 RCF for 10 min) followed by a brief period of incubation was sufficient for the preparation of an active extract to support Cell-Free Protein Synthesis with higher productivity and consistency. Compared to the present standard procedures for the preparation of the S30 extract, the overall cost of the reagents and processing time were reduced by 80 and 60%, respectively.

Osamu Nishimura - One of the best experts on this subject based on the ideXlab platform.

  • Protein prenylation in an insect cell free Protein Synthesis system and identification of products by mass spectrometry
    Proteomics, 2007
    Co-Authors: Masaaki Ito, Toshihiko Utsumi, Eiji Ando, Susumu Tsunasawa, Toru Ezure, Masamitsu Shikata, Osamu Nishimura
    Abstract:

    To evaluate the ability of an insect Cell-Free Protein Synthesis system to carry out proper Protein prenylation, several CAIX (X indicates any C-terminal amino acid) sequences were introduced into the C-terminus of truncated human gelsolin (tGelsolin). Tryptic digests of these mutant Proteins were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The results indicated that the insect Cell-Free Protein Synthesis system possesses both farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) I, as is the case of the rabbit reticulocyte lysate system. The C-terminal amino acid sequence requirements for Protein prenylation in this system showed high similarity to those observed in rat prenyltransferases. In the case of rhoC, which is a natural geranylgeranylated Protein, it was found that it could serve as a substrate for both prenyltransferases in the presence of either farnesyl or geranylgeranyl pyrophosphate, whereas geranylgeranylation was only observed when both prenyl pyrophosphates were added to the in vitro translation reaction mixture. Thus, a combination of the Cell-Free Protein Synthesis system with MS is an effective strategy to analyze Protein prenylation.

  • n terminal Protein modifications in an insect cell free Protein Synthesis system and their identification by mass spectrometry
    Proteomics, 2006
    Co-Authors: Takashi Suzuki, Toshihiko Utsumi, Eiji Ando, Susumu Tsunasawa, Toru Ezure, Masamitsu Shikata, Osamu Nishimura, Masaaki Ito
    Abstract:

    To evaluate the ability of an- insect Cell-Free Protein Synthesis system to generate proper N-terminal cotranslational Protein modifications such as removal of the initiating Met, N-acetylation, and N-myristoylation, several mutants were constructed using truncated human gelsolin (tGelsolin) as a model Protein. Tryptic digests of these mutants were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The wild-type tGelsolin, which is an N-myristoylated Protein, was found to be N-myristoylated when myristoyl-CoA was added to the in vitro translation reaction mixture. N-myristoylation did not occur on the Gly-2 to Ala mutant, in which the N-myristoylation motif was disrupted, whereas this mutant was found to be N-acetylated after removal of the initiating Met. Analyses of Gly-2 to His and Leu-3 to Asp mutants revealed that the amino acids at positions 2 and 3 strongly affect the susceptibility of the nascent peptide chain to removal of the initiating Met and to N-acetylation, respectively. These results suggest that N-terminal modifications occurring in the insect Cell-Free Protein Synthesis system are quite similar to those observed in the mammalian Protein Synthesis system. Thus, a combination of the Cell-Free Protein Synthesis system with MS is an effective strategy to analyze Protein modifications.

  • cell free Protein Synthesis system prepared from insect cells by freeze thawing
    Biotechnology Progress, 2006
    Co-Authors: Toru Ezure, Shoken Higashide, Eiichi Shintani, Kohki Endo, Shinichiro Kobayashi, Masamitsu Shikata, Koji Tanimizu, Osamu Nishimura
    Abstract:

    : We established a novel Cell-Free Protein Synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and beta-galactosidase were synthesized in this system with active forms. We analyzed and optimized (1) the preparation method of the insect cell extract, (2) the concentration of the reaction components, and (3) the 5'-untranslated region (5'-UTR) of mRNA. The extract was prepared by freeze-thawing insect cells suspended in the extraction buffer. This preparation method was a simple and superior method compared with the conventional method using a Dounce homogenizer. Furthermore, Protein Synthesis efficiency was improved by the addition of 20% (v/v) glycerol to the extraction buffer. Concentrations of the reaction components were optimized to increase Protein Synthesis efficiency. Moreover, mRNAs containing 5'-UTRs derived from baculovirus polyhedrin genes showed high Protein Synthesis activity. Especially, the leader composition of the Ectropis obliqua nucleopolyhedrovirus polyhedrin gene showed the highest enhancement activity among the six 5'-UTRs tested. As a result, in a batch reaction approximately 71 microg of luciferase was synthesized per milliliter of reaction volume at 25 degrees C for 6 h. Moreover, this method for the establishment of a Cell-Free system was applied also to Spodoptera frugiperda 21 (Sf21) insect cells. After optimizing the concentrations of the reaction components and the 5'-UTR of mRNA, approximately 45 microg/mL of luciferase was synthesized in an Sf21 Cell-Free system at 25 degrees C for 3 h. These productivities were sufficient to perform gene expression analyses. Thus, these Cell-Free systems may be a useful tool for simple Synthesis in post-genomic studies as a novel Protein production method.

Bradley C Bundy - One of the best experts on this subject based on the ideXlab platform.

  • Protein Synthesis directly from pcr progress and applications of cell free Protein Synthesis with linear dna
    New Biotechnology, 2016
    Co-Authors: Songmin Schinn, Andrew Broadbent, William T Bradley, Bradley C Bundy
    Abstract:

    A rapid, versatile method of Protein expression and screening can greatly facilitate the future development of therapeutic biologics, proteomic drug targets and biocatalysts. An attractive candidate is Cell-Free Protein Synthesis (CFPS), a cell-lysate-based in vitro expression system, which can utilize linear DNA as expression templates, bypassing time-consuming cloning steps of plasmid-based methods. Traditionally, such linear DNA expression templates (LET) have been vulnerable to degradation by nucleases present in the cell lysate, leading to lower yields. This challenge has been significantly addressed in the recent past, propelling LET-based CFPS as a useful tool for studying, screening and engineering Proteins in a high-throughput manner. Currently, LET-based CFPS has promise in fields such as functional proteomics, Protein microarrays, and the optimization of complex biological systems.

  • cell free Protein Synthesis of a cytotoxic cancer therapeutic onconase production and a just add water cell free system
    Biotechnology Journal, 2016
    Co-Authors: Amin S M Salehi, Mark T Smith, Anthony Bennett, Jacob B Williams, William G Pitt, Bradley C Bundy
    Abstract:

    Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. For example, difficult-to-express Proteins may be cytotoxic or form inclusion bodies during expression, increasing the time, labor, and difficulty of purification and downstream characterization. One potential pathway to simplify the expression and screening of such therapeutics is to utilize Cell-Free Protein Synthesis. Cell-Free systems offer a compelling alternative to in vivo production, due to their open and malleable reaction environments. In this work, we demonstrate the use of Cell-Free systems for the expression and direct screening of the difficult-to-express cytotoxic Protein onconase. Using Cell-Free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. This lyophilized system remains viable after being stored above freezing for up to one year. The beneficial features of these Cell-Free systems make them compelling candidates for future biotherapeutic screening and production.

  • lyophilized escherichia coli based cell free systems for robust high density long term storage
    BioTechniques, 2014
    Co-Authors: Mark T Smith, Scott D Berkheimer, Christopher James Werner, Bradley C Bundy
    Abstract:

    Cell-Free Protein Synthesis (CFPS) is a versatile tool for rapid recombinant Protein production and engineering. One drawback of Cell-Free technology is the necessity to store the major components—...

  • streamlined extract preparation for escherichia coli based cell free Protein Synthesis by sonication or bead vortex mixing
    BioTechniques, 2012
    Co-Authors: Prashanta Shrestha, Troy Michael Holland, Bradley C Bundy
    Abstract:

    Escherichia coli-based cell extract is a vital component of inexpensive and high-yielding Cell-Free Protein Synthesis reactions. However, effective preparation of E. coli cell extract is limited to high-pressure (French press-style or impinge-style) or bead mill homogenizers, which all require a significant capital investment. Here we report the viability of E. coli cell extract prepared using equipment that is both common to biotechnology laboratories and able to process small volume samples. Specifically, we assessed the low-capital-cost lysis techniques of: (i) sonication, (ii) bead vortex mixing, (iii) freeze-thaw cycling, and (iv) lysozyme incubation to prepare E. coli cell extract for Cell-Free Protein Synthesis (CFPS). We also used simple shake flask fermentations with a commercially available E. coli strain. In addition, RNA polymerase was overexpressed in the E. coli cells prior to lysis, thus eliminating the need to add independently purified RNA polymerase to the CFPS reaction. As a result, high-yielding E. coli-based extract was prepared using equipment requiring a reduced capital investment and common to biotechnology laboratories. To our knowledge, this is the first successful prokaryote-based CFPS reaction to be carried out with extract prepared by sonication or bead vortex mixing.