The Experts below are selected from a list of 187773 Experts worldwide ranked by ideXlab platform

Hamilton O Smith - One of the best experts on this subject based on the ideXlab platform.

  • enzymatic assembly of dna molecules up to several hundred kilobases
    Nature Methods, 2009
    Co-Authors: Daniel G Gibson, Lei Young, Rayyuan Chuang, Craig J Venter, Clyde A Hutchison, Hamilton O Smith
    Abstract:

    We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular Engineering Tool.

  • enzymatic assembly of dna molecules up to several hundred kilobases
    Nature Methods, 2009
    Co-Authors: Daniel G Gibson, Lei Young, Rayyuan Chuang, Craig J Venter, Clyde A Hutchison, Hamilton O Smith
    Abstract:

    The combination of 5′ exonuclease, DNA polymerase and ligase with overlapping DNA fragments facilitates the in vitro assembly of large DNA constructs, including an entire bacterial genome. We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular Engineering Tool.

Daniel G Gibson - One of the best experts on this subject based on the ideXlab platform.

  • enzymatic assembly of dna molecules up to several hundred kilobases
    Nature Methods, 2009
    Co-Authors: Daniel G Gibson, Lei Young, Rayyuan Chuang, Craig J Venter, Clyde A Hutchison, Hamilton O Smith
    Abstract:

    We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular Engineering Tool.

  • enzymatic assembly of dna molecules up to several hundred kilobases
    Nature Methods, 2009
    Co-Authors: Daniel G Gibson, Lei Young, Rayyuan Chuang, Craig J Venter, Clyde A Hutchison, Hamilton O Smith
    Abstract:

    The combination of 5′ exonuclease, DNA polymerase and ligase with overlapping DNA fragments facilitates the in vitro assembly of large DNA constructs, including an entire bacterial genome. We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular Engineering Tool.

Igor Tascon - One of the best experts on this subject based on the ideXlab platform.

  • segmental isotopic labeling of a single domain globular protein without any refolding step by an asparaginyl endopeptidase
    FEBS Letters, 2017
    Co-Authors: Kornelia M. Mikula, Igor Tascon, Jenni Tommila, Hideo Iwai
    Abstract:

    Asparaginyl endopeptidases (AEPs) catalyze head-to-tail backbone cyclization of naturally occurring cyclic peptides such as cyclotides, and have become an important peptide-Engineering Tool for macrocyclization and peptide ligation. Here, we report efficient protein ligation in trans by mimicking efficient backbone cyclization by an AEP without any excess of reactants. We demonstrate a practical application of segmental isotopic labeling for NMR studies of a single-domain globular protein without any refolding step using the recombinant AEP prepared from Escherichia coli. This simple protein ligation approach using an AEP could be applied for incorporation of various biophysical probes into proteins as well as post-translational production of full-length proteins.

  • salt inducible protein splicing in cis and trans by inteins from extremely halophilic archaea as a novel protein Engineering Tool
    Journal of Molecular Biology, 2016
    Co-Authors: Annika Ciragan, Sesilja A Aranko, Igor Tascon
    Abstract:

    Intervening protein sequences (inteins) from extremely halophilic haloarchaea can be inactive under low salinity but could be activated by increasing the salt content to a specific concentration for each intein. The halo-obligatory inteins confer high solubility under both low and high salinity conditions. We showed the broad utility of salt-dependent protein splicing in cis and trans by demonstrating backbone cyclization, self-cleavage for purification, and scarless protein ligation for segmental isotopic labeling. Artificially split MCM2 intein derived from Halorhabdus utahensis remained highly soluble and was capable of protein trans-splicing with excellent ligation kinetics by reassembly under high salinity conditions. Importantly, the MCM2 intein has the active site residue of Ser at the +1 position, which remains in the ligated product, instead of Cys as found in many other efficient split inteins. Since Ser is more abundant than Cys in proteins, the novel split intein could widen the applications of segmental labeling in protein NMR spectroscopy and traceless protein ligation by exploiting a Ser residue in the native sequences as the +1 position of the MCM2 intein. The split halo-obligatory intein was successfully used to demonstrate the utility in NMR investigation of intact proteins by producing segmentally isotope-labeled intact TonB protein from Helicobacter pylori.

Gerald Weber - One of the best experts on this subject based on the ideXlab platform.

Lei Young - One of the best experts on this subject based on the ideXlab platform.

  • enzymatic assembly of dna molecules up to several hundred kilobases
    Nature Methods, 2009
    Co-Authors: Daniel G Gibson, Lei Young, Rayyuan Chuang, Craig J Venter, Clyde A Hutchison, Hamilton O Smith
    Abstract:

    We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular Engineering Tool.

  • enzymatic assembly of dna molecules up to several hundred kilobases
    Nature Methods, 2009
    Co-Authors: Daniel G Gibson, Lei Young, Rayyuan Chuang, Craig J Venter, Clyde A Hutchison, Hamilton O Smith
    Abstract:

    The combination of 5′ exonuclease, DNA polymerase and ligase with overlapping DNA fragments facilitates the in vitro assembly of large DNA constructs, including an entire bacterial genome. We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular Engineering Tool.