scholarly journals Improving genomically recoded Escherichia coli for the production of proteins containing non-canonical amino acids

2021 ◽  
Author(s):  
Jessica G. Perez ◽  
Erik D. Carlson ◽  
Oliver Weisser ◽  
Camila Kofman ◽  
Kosuke Seki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Production ◽  
Fluorescent Protein ◽  
Cellular Protein ◽  
Release Factor ◽  
Wild Type ◽  
Genetic Encoding ◽  
Lysine Residues ◽  
Green Fluorescent

AbstractA genomically recoded Escherichia coli strain that lacks all amber codons and release factor 1 (C321.ΔA) enables efficient genetic encoding of chemically diverse, non-canonical amino acids (ncAAs) into proteins. While C321.ΔA has opened new opportunities in chemical and synthetic biology, this strain has not been optimized for protein production, limiting its utility in widespread industrial and academic applications. To address this limitation, we describe the construction of a series of genomically recoded organisms that are optimized for cellular protein production. We demonstrate that the functional deactivation of nucleases (e.g., rne, endA) and proteases (e.g., lon) increases production of wild-type superfolder green fluorescent protein (sfGFP) and sfGFP containing two ncAAs up to ∼5-fold. Additionally, we introduce a genomic IPTG-inducible T7 RNA polymerase (T7RNAP) cassette into these strains. Using an optimized platform, we demonstrated the ability to introduce 2 identical N6-(propargyloxycarbonyl)-L-Lysine residues site specifically into sfGFP with a 17-fold improvement in production relative to the parent. We envision that our library of organisms will provide the community with multiple options for increased expression of proteins with new and diverse chemistries.

scholarly journals The sustainability of interactions between the orexin-1 receptor and β-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation

2005 ◽  
Vol 387 (3) ◽  
pp. 573-584 ◽  
Author(s):  
Sandra MILASTA ◽  
Nicholas A. EVANS ◽  
Laura ORMISTON ◽  
Shelagh WILSON ◽  
Robert J. LEFKOWITZ ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Weak Interactions ◽  
Dependent Manner ◽  
Wild Type ◽  
Erk Mapk ◽  
Hydroxy Amino ◽  
C Terminus ◽  
Green Fluorescent ◽  
Kinetics Of

The orexin-1 receptor interacts with β-arrestin-2 in an agonist-dependent manner. In HEK-293T cells, these two proteins became co-internalized into acidic endosomes. Truncations from the C-terminal tail did not prevent agonist-induced internalization of the orexin-1 receptor or alter the pathway of internalization, although such mutants failed to interact with β-arrestin-2 in a sustained manner or produce its co-internalization. Mutation of a cluster of three threonine and one serine residue at the extreme C-terminus of the receptor greatly reduced interaction and abolished co-internalization of β-arrestin-2–GFP (green fluorescent protein). Despite the weak interactions of this C-terminally mutated form of the receptor with β-arrestin-2, studies in wild-type and β-arrestin-deficient mouse embryo fibroblasts confirmed that agonist-induced internalization of this mutant required expression of a β-arrestin. Although without effect on agonist-mediated elevation of intracellular Ca2+ levels, the C-terminally mutated form of the orexin-1 receptor was unable to sustain phosphorylation of the MAPKs (mitogen-activated protein kinases) ERK1 and ERK2 (extracellular-signal-regulated kinases 1 and 2) to the same extent as the wild-type receptor. These studies indicate that a single cluster of hydroxy amino acids within the C-terminal seven amino acids of the orexin-1 receptor determine the sustainability of interaction with β-arrestin-2, and indicate an important role of β-arrestin scaffolding in defining the kinetics of orexin-1 receptor-mediated ERK MAPK activation.

scholarly journals mhpTEncodes an Active Transporter Involved in 3-(3-Hydroxyphenyl)Propionate Catabolism by Escherichia coli K-12

2013 ◽  
Vol 79 (20) ◽  
pp. 6362-6368 ◽  
Author(s):  
Ying Xu ◽  
Bing Chen ◽  
Hongjun Chao ◽  
Ning-Yi Zhou
Keyword(s):  
Escherichia Coli ◽  
Wild Type Strain ◽  
Fluorescent Protein ◽  
Gene Knockout ◽  
Transport Activity ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent ◽  
K 12

ABSTRACTEscherichia coliK-12 utilizes 3-(3-hydroxyphenyl)propionate (3HPP) as a sole carbon and energy source. Among the genes in its catabolic cluster in the genome,mhpTwas proposed to encode a hypothetical transporter. Since no transporter for 3HPP uptake has been identified, we investigated whether MhpT is responsible for 3HPP uptake. MhpT fused with green fluorescent protein was found to be located at the periphery of cells by confocal microscopy, consistent with localization to the cytoplasmic membrane. Gene knockout and complementation studies clearly indicated thatmhpTis essential for 3HPP catabolism inE. coliK-12 W3110 at pH 8.2. Uptake assays with14C-labeled substrates demonstrated that strain W3110 and strain W3110ΔmhpTcontaining recombinant MhpT specifically transported 3HPP but not benzoate, 3-hydroxybenzoate, or gentisate into cells. Energy dependence assays suggested that MhpT-mediated 3HPP transport was driven by the proton motive force. The change of Ala-272 of MhpT to a histidine, surprisingly, resulted in enhanced transport activity, and strain W3110ΔmhpTcontaining the MhpT A272H mutation had a slightly higher growth rate than the wild-type strain at pH 8.2. Hence, we demonstrated that MhpT is a specific 3HPP transporter and vital forE. coliK-12 W3110 growth on this substrate under basic conditions.

scholarly journals Translation enhancement by a Dictyostelium gene sequence in Escherichia coli

2019 ◽  
Author(s):  
Tomo Kondo ◽  
Shigehiko Yumura
Keyword(s):  
Escherichia Coli ◽  
Protein Production ◽  
Gene Sequence ◽  
Fluorescent Protein ◽  
Production Systems ◽  
Expression System ◽  
Protein Product ◽  
A Subunit ◽  
Shine Dalgarno Sequence ◽  
Green Fluorescent

AbstractMethods for heterologous protein production in Escherichia coli have revolutionized biotechnology and the bioindustry. It is ultimately important to increase the amount of protein product from bacteria. To this end, a variety of tools, such as effective promoters, have been developed. Here, we present a versatile molecular tool based on a phenomenon termed “translation enhancement by a Dictyostelium gene sequence” (“TED”) in E. coli. We found that protein expression was increased when a gene sequence of Dictyostelium discoideum was placed upstream of the Shine-Dalgarno sequence located between the promoter and the initiation codon of a target gene. The most effective sequence among the genes examined was mlcR, which encodes the myosin regulatory light chain, a subunit of myosin II. Serial deletion analysis revealed that at least 10 bases of the 3’ end of the mlcR gene enhanced the production of green fluorescent protein in cells. We applied this tool to a T7 expression system and found that the expression level of the proteins tested was increased when compared with the conventional method. Thus, current protein production systems can be improved by combination with TED.

scholarly journals Polyhydroxyalkanoate Inclusion Body-Associated Proteins and Coding Region in Bacillus megaterium

1999 ◽  
Vol 181 (2) ◽  
pp. 585-592 ◽  
Author(s):  
Gabriel J. McCool ◽  
Maura C. Cannon
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Inclusion Body ◽  
Bacillus Megaterium ◽  
Inclusion Bodies ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Coding Region ◽  
Associated Proteins ◽  
Green Fluorescent

ABSTRACT Polyhydroxyalkanoic acids (PHA) are carbon and energy storage polymers that accumulate in inclusion bodies in many bacteria and archaea in response to environmental conditions. This work presents the results of a study of PHA inclusion body-associated proteins and an analysis of their coding region in Bacillus megaterium 11561. A 7,917-bp fragment of DNA was cloned and shown to carry a 4,104-bp cluster of 5 pha genes,phaP, -Q, -R, -B, and -C. The phaP and -Q genes were shown to be transcribed in one orientation, each from a separate promoter, while immediately upstream, phaR, -B, and -C were divergently transcribed as a tricistronic operon. Transfer of this gene cluster to Escherichia coliand to a PhaC− mutant of Pseudomonas putidagave a Pha+ phenotype in both strains. Translational fusions to the green fluorescent protein localized PhaP and PhaC to the PHA inclusion bodies in living cells. The data presented are consistent with the hypothesis that the extremely hydrophilic protein PhaP is a storage protein and suggests that PHA inclusion bodies are not only a source of carbon, energy, and reducing equivalents but are also a source of amino acids.

scholarly journals Presence of Multiple Sites Containing Polar Material in Spherical Escherichia coli Cells That Lack MreB

2005 ◽  
Vol 187 (17) ◽  
pp. 6187-6196 ◽  
Author(s):  
Trine Nilsen ◽  
Arthur W. Yan ◽  
Gregory Gale ◽  
Marcia B. Goldberg
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Positional Information ◽  
Actin Assembly ◽  
Wild Type ◽  
Polar Material ◽  
Transposon Insertion ◽  
Escherichia Coli Cells ◽  
Inner Membrane Protein ◽  
Green Fluorescent

ABSTRACT In rod-shaped bacteria, certain proteins are specifically localized to the cell poles. The nature of the positional information that leads to the proper localization of these proteins is unclear. In a screen for factors required for the localization of the Shigella sp. actin assembly protein IcsA to the bacterial pole, a mutant carrying a transposon insertion in mreB displayed altered targeting of IcsA. The phenotype of cells containing a transposon insertion in mreB was indistinguishable from that of cells containing a nonpolar mutation in mreB or that of wild-type cells treated with the MreB inhibitor A22. In cells lacking MreB, a green fluorescent protein (GFP) fusion to a cytoplasmic derivative of IcsA localized to multiple sites. Secreted full-length native IcsA was present in multiple faint patches on the surfaces of these cells in a pattern similar to that seen for the cytoplasmic IcsA-GFP fusion. EpsM, the polar Vibrio cholerae inner membrane protein, also localized to multiple sites in mreB cells and colocalized with IcsA, indicating that localization to multiple sites is not unique to IcsA. Our results are consistent with the requirement, either direct or indirect, for MreB in the restriction of certain polar material to defined sites within the cell and, in the absence of MreB, with the formation of ectopic sites containing polar material.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

scholarly journals Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

2005 ◽  
Vol 25 (12) ◽  
pp. 4977-4992 ◽  
Author(s):  
Hao G. Nguyen ◽  
Dharmaraj Chinnappan ◽  
Takeshi Urano ◽  
Katya Ravid
Keyword(s):  
Chromosome Segregation ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Degradation Pathway ◽  
Aurora B ◽  
Stable Form ◽  
Wild Type ◽  
Green Fluorescent ◽  
Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

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