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Träfflista för sökning "WFRF:(Nørholm Morten H. H.) "

Sökning: WFRF:(Nørholm Morten H. H.)

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1.
  • Baumgarten, Thomas, et al. (författare)
  • Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)
  • 2017
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
  • Tidskriftsartikel (refereegranskat)abstract
    • Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3) changes one amino acid in its T7 RNAP, which weakens the binding of the T7 RNAP to the T7 promoter governing target gene expression rather than lowering T7 RNAP levels. For most membrane proteins tested yields in Mutant56(DE3) were considerably higher than in C41(DE3) and C43(DE3). Thus, the isolation of Mutant56(DE3) shows that the evolution of BL21(DE3) can be promoted towards further enhanced membrane protein production.
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2.
  • Brander, Søren, et al. (författare)
  • Biochemical evidence of both copper chelation and oxygenase activity at the histidine brace
  • 2020
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Lytic polysaccharide monooxygenase (LPMO) and copper binding protein CopC share a similar mononuclear copper site. This site is defined by an N-terminal histidine and a second internal histidine side chain in a configuration called the histidine brace. To understand better the determinants of reactivity, the biochemical and structural properties of a well-described cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A) is compared with that of CopC from Pseudomonas fluorescens (PfCopC) and with the LPMO-like protein Bim1 from Cryptococcus neoformans. PfCopC is not reduced by ascorbate but is a very strong Cu(II) chelator due to residues that interacts with the N-terminus. This first biochemical characterization of Bim1 shows that it is not redox active, but very sensitive to H2O2, which accelerates the release of Cu ions from the protein. TaAA9A oxidizes ascorbate at a rate similar to free copper but through a mechanism that produce fewer reactive oxygen species. These three biologically relevant examples emphasize the diversity in how the proteinaceous environment control reactivity of Cu with O2.
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3.
  • Cumming, Alister James, et al. (författare)
  • Antibiotic-Efficient Genetic Cassette for the TEM-1 β-Lactamase That Improves Plasmid Performance
  • 2022
  • Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 11:1, s. 241-253
  • Tidskriftsartikel (refereegranskat)abstract
    • Antibiotic resistance cassettes are indispensable tools in recombinant DNA technology, synthetic biology, and metabolic engineering. The genetic cassette encoding the TEM-1 β-lactamase (denoted Tn3.1) is one of the most commonly used and can be found in more than 120 commercially available bacterial expression plasmids (e.g., the pET, pUC, pGEM, pQE, pGEX, pBAD, and pSEVA series). A widely acknowledged problem with the cassette is that it produces excessively high titers of β-lactamase that rapidly degrade β-lactam antibiotics in the culture media, leading to loss of selective pressure, and eventually a large percentage of cells that do not have a plasmid. To address these shortcomings, we have engineered a next-generation version that expresses minimal levels of β-lactamase (denoted Tn3.1MIN). We have also engineered a version that is compatible with the Standard European Vector Architecture (SEVA) (denoted Ap (pSEVA#1MIN--)). Expression plasmids containing either Tn3.1MIN or Ap (pSEVA#1MIN--) can be selected using a 5-fold lower concentration of β-lactam antibiotics and benefit from the increased half-life of the β-lactam antibiotics in the culture medium (3- to 10-fold). Moreover, more cells in the culture retain the plasmid. In summary, we present two antibiotic-efficient genetic cassettes encoding the TEM-1 β-lactamase that reduce antibiotic consumption (an integral part of antibiotic stewardship), reduce production costs, and improve plasmid performance in bacterial cell factories. 
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4.
  • Ferro, Roberto, et al. (författare)
  • A synbio approach for selection of highly expressed gene variants in Gram-positive bacteria
  • 2018
  • Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 17
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: The market for recombinant proteins is on the rise, and Gram-positive strains are widely exploited for this purpose. Bacillus subtilis is a profitable host for protein production thanks to its ability to secrete large amounts of proteins, and Lactococcus lactis is an attractive production organism with a long history in food fermentation. Results: We have developed a synbio approach for increasing gene expression in two Gram-positive bacteria. First of all, the gene of interest was coupled to an antibiotic resistance gene to create a growth-based selection system. We then randomised the translation initiation region (TIR) preceding the gene of interest and selected clones that produced high protein titres, as judged by their ability to survive on high concentrations of antibiotic. Using this approach, we were able to significantly increase production of two industrially relevant proteins; sialidase in B. subtilis and tyrosine ammonia lyase in L. lactis. Conclusion: Gram-positive bacteria are widely used to produce industrial enzymes. High titres are necessary to make the production economically feasible. The synbio approach presented here is a simple and inexpensive way to increase protein titres, which can be carried out in any laboratory within a few days. It could also be implemented as a tool for applications beyond TIR libraries, such as screening of synthetic, homologous or domain-shuffled genes.
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5.
  • Hernández-Rollán, Cristina, et al. (författare)
  • LyGo : A Platform for Rapid Screening of Lytic Polysaccharide Monooxygenase Production
  • 2021
  • Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 10:4, s. 897-906
  • Tidskriftsartikel (refereegranskat)abstract
    • Environmentally friendly sources of energy and chemicals are essential constituents of a sustainable society. An important step toward this goal is the utilization of biomass to supply building blocks for future biorefineries. Lytic polysaccharide monooxygenases (LPMOs) are enzymes that play a critical role in breaking the chemical bonds in the most abundant polymers found in recalcitrant biomass, such as cellulose and chitin. To use them in industrial processes they need to be produced in high titers in cell factories. Predicting optimal strategies for producing LPMOs is often nontrivial, and methods allowing for screening several strategies simultaneously are therefore needed. Here, we present a standardized platform for cloning LPMOs. The platform allows users to combine gene fragments with 14 different expression vectors in a simple 15 min reaction, thus enabling rapid exploration of several gene contexts, hosts, and expression strategies in parallel. The open-source LyGo platform is accompanied by easy-to-follow online protocols for both cloning and expression. As a demonstration of its utility, we explore different strategies for expressing several different LPMOs in Escherichia coli, Bacillus subtilis, and Komagataella phaffii.
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6.
  • Mirzadeh, Kiavash, et al. (författare)
  • Codon Optimizing for Increased Membrane Protein Production : A Minimalist Approach
  • 2016
  • Ingår i: Heterologous Expression of Membrane Proteins. - New York : Humana Press. - 9781493936359 - 9781493936373 ; , s. 53-61
  • Bokkapitel (refereegranskat)abstract
    • Reengineering a gene with synonymous codons is a popular approach for increasing production levels of recombinant proteins. Here we present a minimalist alternative to this method, which samples synonymous codons only at the second and third positions rather than the entire coding sequence. As demonstrated with two membrane-embedded transporters in Escherichia coli, the method was more effective than optimizing the entire coding sequence. The method we present is PCR based and requires three simple steps: (1) the design of two PCR primers, one of which is degenerate; (2) the amplification of a mini-library by PCR; and (3) screening for high-expressing clones.
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7.
  • Mirzadeh, Kiavash, et al. (författare)
  • Efficient protein production requires selection at the vector : coding sequence junction
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Here we show that the junction formed when a coding sequence is cloned into an expression vector can affect protein production levels by up to a 1000-fold. Optimised junctions can be identified after a post-cloning optimisation step, which generates a library of sequence variants that differ only in ribosome-binding sites. The approach is simple, inexpensive and applicable to any experiment where efficient expression of a cloned coding sequence is sought.
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8.
  • Mirzadeh, Kiavash, et al. (författare)
  • Enhanced Protein Production in Escherichia coli by Optimization of Cloning Scars at the Vector-Coding Sequence Junction
  • 2015
  • Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 4:9, s. 959-965
  • Tidskriftsartikel (refereegranskat)abstract
    • Protein production in Escherichia coli is a fundamental activity for a large fraction of academic, pharmaceutical, and industrial research laboratories. Maximum production is usually sought, as this reduces costs and facilitates downstream purification steps. Frustratingly, many coding sequences are poorly expressed even when they are codon-optimized and expressed from vectors with powerful genetic elements. In this study, we show that poor expression can be caused by certain nucleotide sequences (e.g., cloning scars) at the junction between the vector and the coding sequence. Since these sequences lie between the Shine-Dalgarno sequence and the start codon, they are an integral part of the translation initiation region. To identify the most optimal sequences, we devised a simple and inexpensive PCR-based step that generates sequence variants at the vector-coding sequence junction. These sequence variants modulated expression by up to WOO-fold. FACS-seq analyses indicated that low GC content and relaxed mRNA stability (AG) in this region were important, but not the only, determinants for high expression.
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9.
  • Mirzadeh, Kiavash, et al. (författare)
  • Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region
  • 2020
  • Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 19:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Recombinant proteins are often engineered with an N-terminal signal peptide, which facilitates their secretion to the oxidising environment of the periplasm (gram-negative bacteria) or the culture supernatant (gram-positive bacteria). A commonly encountered problem is that the signal peptide influences the synthesis and secretion of the recombinant protein in an unpredictable manner. A molecular understanding of this phenomenon is highly sought after, as it could lead to improved methods for producing recombinant proteins in bacterial cell factories.Results: Herein we demonstrate that signal peptides contribute to an unpredictable translation initiation region. A directed evolution approach that selects a new translation initiation region, whilst leaving the amino acid sequence of the signal peptide unchanged, can increase production levels of secreted recombinant proteins. The approach can increase production of single chain antibody fragments, hormones and other recombinant proteins in the periplasm of E. coli.Conclusions: The study demonstrates that signal peptide performance is coupled to the efficiency of the translation initiation region.
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10.
  • Nørholm, Morten H. H. (författare)
  • A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering
  • 2010
  • Ingår i: BMC Biotechnology. - : Springer Science and Business Media LLC. - 1472-6750. ; 10, s. 21-27
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: The combined use of restriction enzymes with PCR has revolutionized molecular cloning, but is inherently restricted by the content of the manipulated DNA sequences. Uracil-excision based cloning is ligase and sequence independent and allows seamless fusion of multiple DNA sequences in simple one-tube reactions, with higher accuracy than overlapping PCR. Results: Here, the addition of a highly efficient DNA polymerase and a low-background-, large-insertion-compatible site-directed mutagenesis protocol is described, largely expanding the versatility of uracil-excision DNA engineering. Conclusions: The different uracil-excision based molecular tools that have been developed in an open-source fashion, constitute a comprehensive, yet simple and inexpensive toolkit for any need in molecular cloning.
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11.
  • Nørholm, Morten H. H., et al. (författare)
  • Converting a Marginally Hydrophobic Soluble Protein into a Membrane Protein
  • 2011
  • Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 407:1, s. 171-179
  • Tidskriftsartikel (refereegranskat)abstract
    • delta-Helices are marginally hydrophobic a-helical segments in soluble proteins that exhibit certain sequence characteristics of transmembrane (TM) helices [Cunningham, F., Rath, A., Johnson, R. M. & Deber, C. M. (2009). Distinctions between hydrophobic helices in globular proteins and TM segments as factors in protein sorting. J. Biol. Chem., 284, 5395-402]. In order to better understand the difference between delta-helices and TM helices, we have studied the insertion of five TM-like delta-helices into dog pancreas microsomal membranes. Using model constructs in which an isolated delta-helix is engineered into a bona fide membrane protein, we find that, for two delta-helices originating from secreted proteins, at least three single-nucleotide mutations are necessary to obtain efficient membrane insertion, whereas one mutation is sufficient in a delta-helix from the cytosolic protein P450BM-3. We further find that only when the entire upstream region of the mutated delta-helix in the intact cytochrome P450BM-3 is deleted does a small fraction of the truncated protein insert into microsomes. Our results suggest that upstream portions of the polypeptide, as well as embedded charged residues, protect delta-helices in globular proteins from being recognized by the signal recognition particle-Sec61 endoplasmic-reticulum-targeting machinery and that delta-helices in secreted proteins are mutationally more distant from TM helices than delta-helices in cytosolic proteins.
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12.
  • Nørholm, Morten H. H., et al. (författare)
  • Flanking Residues Help Determine Whether a Hydrophobic Segment Adopts a Monotopic or Bitopic Topology in the Endoplasmic Reticulum Membrane
  • 2011
  • Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 286:28, s. 25284-25290
  • Tidskriftsartikel (refereegranskat)abstract
    • Proteins interacting with membranes via a single hydrophobic segment can be classified as either monotopic or bitopic. Here, we probe the topology of a membrane-attached enzyme, the epsilon isoform of human diacylglycerol kinase (DGK epsilon), when inserted into rough microsomes and compare it with the monotopic membrane protein mouse caveolin-1. In contrast to previous findings, the N-terminal hydrophobic stretch in DGK epsilon attains a bitopic rather than a monotopic topology in our experimental system. In addition, we find that charged flanking residues as well as proline residues embedded in the hydrophobic segment are important determinants of monotopic versus bitopic topology.
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13.
  • Nørholm, Morten H. H., et al. (författare)
  • Manipulating the genetic code for membrane protein production : What have we learnt so far?
  • 2012
  • Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0005-2736. ; 1818:4, s. 1091-1096
  • Forskningsöversikt (refereegranskat)abstract
    • With synthetic gene services, molecular cloning is as easy as ordering a pizza. However choosing the right RNA code for efficient protein production is less straightforward, more akin to deciding on the pizza toppings. The possibility to choose synonymous codons in the gene sequence has ignited a discussion that dates back 50years: Does synonymous codon use matter? Recent studies indicate that replacement of particular codons for synonymous codons can improve expression in homologous or heterologous hosts, however it is not always successful. Furthermore it is increasingly apparent that membrane protein biogenesis can be codon-sensitive. Single synonymous codon substitutions can influence mRNA stability, mRNA structure, translational initiation, translational elongation and even protein folding. Synonymous codon substitutions therefore need to be carefully evaluated when membrane proteins are engineered for higher production levels and further studies are needed to fully understand how to select the codons that are optimal for higher production. This article is part of a Special Issue entitled: Protein Folding in Membranes.
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14.
  • Rennig, Maja, et al. (författare)
  • TARSyn : Tunable Antibiotic Resistance Devices Enabling Bacterial Synthetic Evolution and Protein Production
  • 2018
  • Ingår i: ACS Photonics. - : American Chemical Society (ACS). - 2330-4022. ; 7:2, s. 432-442
  • Tidskriftsartikel (refereegranskat)abstract
    • Evolution can be harnessed to optimize synthetic biology designs. A prominent example is recombinant protein production-a dominating theme in biotechnology for more than three decades. Typically, a protein coding sequence (cds) is recombined with genetic elements, such as promoters, ribosome binding sites and terminators, which control expression in a cell factory. A major bottleneck during production is translational initiation. Previously we identified more effective translation initiation regions (TIRs) by creating sequence libraries and then selecting for a TIR that drives high-level expression-an example of synthetic evolution. However, manual screening limits the ability to assay expression levels of all putative sequences in the libraries. Here we have solved this bottleneck by designing a collection of translational coupling devices based on a RNA secondary structure. Exchange of different sequence elements in this device allows for different coupling efficiencies, therefore giving the devices a tunable nature. Sandwiching these devices between the cds and an antibiotic selection marker that functions over a broad dynamic range of antibiotic concentrations adds to the tunability and allows expression levels in large clone libraries to be probed using a simple cell survival assay on the respective antibiotic. The power of the approach is demonstrated by substantially increasing production of two commercially interesting proteins, a Nanobody and an Affibody. The method is a simple and inexpensive alternative to advanced screening techniques that can be carried out in any laboratory.
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