| 1. |
- Wang, Harris H., et al.
(author)
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Multiplexed in Vivo His-Tagging of Enzyme Pathways for in Vitro Single-Pot Multienzyme Catalysis
- 2012
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In: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 1:2, s. 43-52
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Journal article (peer-reviewed)abstract
- Protein pathways are dynamic and highly coordinated spatially and temporally, capable of performing a diverse range of complex chemistries and enzymatic reactions with precision and at high efficiency. Biotechnology aims to harvest these natural systems to construct more advanced in vitro reactions, capable of new chemistries and operating at high yield. Here, we present an efficient Multiplex Automated Genome Engineering (MAGE) strategy to simultaneously modify and co-purify large protein complexes and pathways from the model organism Escherichia coli to reconstitute functional synthetic proteomes in vitro. By application of over 110 MAGE cycles, we successfully inserted hexa-histidine sequences into 38 essential genes in vivo that encode for the entire translation machinery. Streamlined co-purification and reconstitution of the translation, protein complex enabled protein synthesis in vitro. Our approach can be applied to a growing area of applications in in vitro one-pot multienzyme catalysis (MEC) to manipulate or enhance in vitro pathways such as natural product or carbohydrate biosynthesis.
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| 2. |
- Bao, Letian, et al.
(author)
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Overcoming chromoprotein limitations by engineering a red fluorescent protein
- 2020
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In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 611
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Journal article (peer-reviewed)abstract
- Chromoproteins (CPs) are widely-used visual reporters of gene expression. We previously showed that, for coloration in Escherichia coli, CPs had to be overexpressed and that this caused large fitness costs with the most useful (darkly colored) CPs. These fitness costs were problematic because passage of plasmids encoding darkly colored CPs in liquid culture frequently resulted in loss of color due to mutations. Unexpectedly, an early variant of the monomeric red fluorescent protein 1 (mRFP1) gene that was codon-optimized for E. coli (abbreviated mRFP1E) was found here to be an ideal replacement for CP genes. When we subcloned mRFP1E in the same way as our CP genes, it produced a similarly dark color, yet affected E. coli fitness minimally. This finding facilitated testing of several hypotheses on the cause of CP cytotoxicities by gel electrophoresis and size-exclusion chromatography: toxicities correlated with the combination of amounts of expression, oligomerization and inclusion bodies, not isoelectric point. Finally, a semi-rational mutagenesis strategy created several mRFP1 protein variants with different colors without altering the fitness cost. Thus, these mutants and mRFP1E are suitable for comparative fitness costs between different strains of E. coli. We conclude that our new mRFP1E series overcomes prior limitations of CPs.
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| 3. |
- Bao, Letian, et al.
(author)
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Rate-limiting hydrolysis in ribosomal release reactions revealed by ester activation
- 2022
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In: Journal of Biological Chemistry. - : Elsevier. - 0021-9258 .- 1083-351X. ; 28:11
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Journal article (peer-reviewed)abstract
- Translation terminates by releasing the polypeptide chain in one of two chemical reactions catalyzed by the ribosome. Release is also a target for engineering, as readthrough of a stop codon enables incorporation of unnatural amino acids and treatment of genetic diseases. Hydrolysis of the ester bond of peptidyl-tRNA requires conformational changes of both a class I release factor (RF) protein and the peptidyl transferase center of a large subunit rRNA. The rate-limiting step was proposed to be hydrolysis at physiological pH and an RF conformational change at higher pH, but evidence was indirect. Here, we tested this by activating the ester electrophile at the Escherichia coli ribosomal P site using a trifluorine-substituted amino acid. Quench-flow kinetics revealed that RF1-catalyzed release could be accelerated, but only at pH 6.2-7.7 and not higher pH. This provided direct evidence for rate-limiting hydrolysis at physiological or lower pH and a different rate limitation at higher pH. Additionally, we optimized RF-free release catalyzed by unacylated tRNA or the CCA trinucleotide (in 30% acetone). We determined that these two model release reactions, although very slow, were surprisingly accelerated by the trifluorine analog but to a different extent from each other and from RF-catalyzed release. Hence, hydrolysis was rate limiting in all three reactions. Furthermore, in 20% ethanol, we found that there was significant competition between fMet-ethyl ester formation and release in all three release reactions. We thus favor proposed mechanisms for translation termination that do not require a fully-negatively-charged OH− nucleophile.
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| 4. |
- Bao, Letian, et al.
(author)
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Translational impacts of enzymes that modify ribosomal RNA around the peptidyl transferase centre
- 2024
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In: RNA Biology. - : Taylor & Francis. - 1547-6286 .- 1555-8584. ; 21:1, s. 31-41
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Journal article (peer-reviewed)abstract
- Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the ‘critical region’ of the peptidyl transferase centre (PTC) in 23S rRNA that exhibited only a minor growth defect at 37°C (although major at 20°C). However, our combined KO of modification enzymes RluC and RlmE (not RluE) resulted in conditional lethality (at 20°C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. In vitro fast kinetics at 20°C and 37°C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Elongation rates of protein synthesis in vivo, as judged by the kinetics of β-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37°C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg2+. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications per se, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.
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| 5. |
- Borgatti, Antonella, et al.
(author)
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Safe and Effective Sarcoma Therapy through Bispecific Targeting of EGFR and uPAR.
- 2017
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In: Molecular Cancer Therapeutics. - 1535-7163 .- 1538-8514. ; 16:5, s. 956-965
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Journal article (peer-reviewed)abstract
- Sarcomas differ from carcinomas in their mesenchymal origin. Therapeutic advancements have come slowly so alternative drugs and models are urgently needed. These studies report a new drug for sarcomas that simultaneously targets both tumor and tumor neovasculature. eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to epidermal growth factor (EGF) and the amino terminal fragment (ATF) of urokinase. Here, we study the drug in an in vivo "ontarget" companion dog trial since eBAT effectively kills canine hemangiosarcoma (HSA) and human sarcoma cells in vitro. We reasoned the model has value due to the common occurrence of spontaneous sarcomas in dogs and a limited lifespan allowing for rapid accrual and data collection. Splenectomized dogs with minimal residual disease were given one cycle of eBAT followed by adjuvant doxorubicin in an adaptive dose-finding, phase I-II study of 23 dogs with spontaneous, stage I-II, splenic HSA. eBAT improved 6-month survival from <40% in a comparison population to ~70% in dogs treated at a biologically active dose (50 µg/kg). Six dogs were long-term survivors, living >450 days. eBAT abated expected toxicity associated with EGFR-targeting, a finding supported by mouse studies. Urokinase plasminogen activator receptor (uPAR) and EGFR are targets for human sarcomas, so thorough evaluation is crucial for validation of the dog model. Thus, we validated these markers for human sarcoma targeting in the study of 212 human and 97 canine sarcoma samples. Our results support further translation of eBAT for human patients with sarcomas and perhaps other EGFR-expressing malignancies.
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| 6. |
- Doerr, Anne, et al.
(author)
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In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity
- 2021
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In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
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Journal article (peer-reviewed)abstract
- The Protein synthesis Using Recombinant Elements (PURE) system enables transcription and translation of a DNA template from purified components. Therefore, the PURE system-catalyzed generation of RNAs and proteins constituting the PURE system itself represents a major challenge toward a self-replicating minimal cell. In this work, we show that all translation factors (except elongation factor Tu) and 20 aminoacyl-tRNA synthetases can be expressed in the PURE system from a single plasmid encoding 32 proteins in 30 cistrons. Cell-free synthesis of all 32 proteins is confirmed by quantitative mass spectrometry-based proteomic analysis using isotopically labeled amino acids. We find that a significant fraction of the gene products consists of proteins missing their C-terminal ends. The per-codon processivity loss that we measure lies between 1.3 x 10(-3) and 13.2 x 10(-3), depending on the expression conditions, the version of the PURE system, and the coding sequence. These values are 5 to 50 times higher than those measured in vivo in E. coli. With such an impaired processivity, a considerable fraction of the biosynthesis capacity of the PURE system is wasted, posing an unforeseen challenge toward the development of a self-regenerating PURE system.
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| 7. |
- Du, Liping, et al.
(author)
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Multigene expression in vivo : Supremacy of large versus small terminators for T7 RNA polymerase
- 2012
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 109:4, s. 1043-1050
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Journal article (peer-reviewed)abstract
- Designing and building multigene constructs is commonplace in synthetic biology. Yet functional successes at first attempts are rare because the genetic parts are not fully modular. In order to improve the modularity of transcription, we previously showed that transcription termination in vitro by bacteriophage T7 RNA polymerase could be made more efficient by substituting the standard, single, TF large (class I) terminator with adjacent copies of the vesicular stomatitis virus (VSV) small (class II) terminator. However, in vitro termination at the downstream VSV terminator was less efficient than at the upstream VSV terminator, and multigene overexpression in vivo was complicated by unexpectedly inefficient VSV termination within Escherichia coli cells. Here, we address hypotheses raised in that study by showing that VSV or preproparathyroid hormone (PTH) small terminators spaced further apart can work independently (i.e., more efficiently) in vitro, and that VSV and PTH terminations are severely inhibited in vivo. Surprisingly, the difference between class II terminator function in vivo versus in vitro is not due to differences in plasmid supercoiling, as supercoiling had a minimal effect on termination in vitro. We therefore turned to TF terminators for BioBrick synthesis of a pentameric gene construct suitable for overexpression in vivo. This indeed enabled coordinated overexpression and copurification of five His-tagged proteins using the first construct attempted, indicating that this strategy is more modular than other strategies. An application of this multigene overexpression and protein copurification method is demonstrated by supplying five of the six E. coli translation factors required for reconstitution of translation from a single cell line via copurification, greatly simplifying the reconstitution.
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| 8. |
- Forster, Anthony C., et al.
(author)
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Editorial : NextGen SynBio has arrived...
- 2012
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In: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 7:7, s. 827-827
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Journal article (other academic/artistic)
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| 9. |
- Forster, Anthony C.
(author)
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Revisiting the Extinction of the RNA World
- 2022
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 61:9, s. 749-751
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Journal article (peer-reviewed)abstract
- The ribozyme world is thought to have evolved the burdensome complexity of peptide and protein synthesis because the 20 amino acid side chains are catalytically superior. Instead, I propose that the Achilles heel of the RNA world that led to the extinction of riboorganisms was RNA's polyanionic charges that could not be covalently neutralized stably by phosphotriester formation. These charges prevented development of hydrophobic cores essential for integration into membranes and many enzymatic reactions. In contrast, the phosphotriester modification of DNA is stable. So, the fact that the charge was never removed in DNA evolution gives further credence to proteins coming before DNA.
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| 10. |
- Forster, Anthony C.
(author)
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Synthetic biology challenges long-held hypotheses in translation, codon bias and transcription
- 2012
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In: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 7:7, s. 835-845
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Research review (peer-reviewed)abstract
- Synthetic biology is a powerful experimental approach, not only for developing new biotechnology applications, but also for testing hypotheses in basic biological science. Here, examples from our research using the best model system, Escherichia coli, are reviewed. New evidence drawn from synthetic biology has overturned several long-standing hypotheses regarding the mechanisms of transcription and translation: (i) all native aminoacyl-tRNAs are not equally efficient in translation at equivalent concentrations; (ii) accommodation is not always rate limiting in translation, and may not be for any aminoacyl-tRNA; (iii) proline is the only N-alkyl-amino acid in the genetic code not because of special suitability for protein structure, but because of its comparatively high nucleophilicity; (iv) the usages of most sense codons in E. coli do not correlate with cognate tRNA abundances and (v) class II transcriptional pausing and termination by T7 RNA polymerase cannot be assumed to occur in vivo based on in vitro data. Implications of these conclusions for the biotechnology field are discussed.
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