Purification and characterization of the L-Ara4N transferase protein ArnT from Salmonella typhimurium.


:The covalent addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) groups to lipid A, which resides in the outer membranes of bacteria such as Salmonella typhimurium and Escherichia coli, is the final step in the polymyxin-resistance pathway in these organisms. This modification is catalyzed by the inner membrane protein 4-amino-4-deoxy-L-arabinose transferase (ArnT). Little is known about the ArnT protein structure because it has not previously been purified. We report here the first expression and purification of 6 x His-tagged S. typhimurium ArnT in NovaBlue cells. The enzyme was purified using sequential Q-Sepharose anion exchange and HisLink nickel affinity column chromatography. The purified protein has an apparent molecular weight of 62 kDa on SDS-PAGE and the identity of the purified ArnT was confirmed by Western blot using a monoclonal antibody against the His-tag and by MALDI-TOF mass spectrometry. Purified ArnT protein was shown to be highly alpha-helical as determined by circular dichroism analysis. A chromosomal ArnT knockout strain of E. coli BL21(DE3) was developed to allow in vivo functional analysis of plasmid-encoded ArnT constructs, and a polymyxin assay was used to confirm that the cloned ArnT proteins retained full activity. These studies provide an essential foundation for further analysis of ArnT structure and function using mutagenesis and biophysical techniques.


Protein Expr Purif


Bretscher LE,Morrell MT,Funk AL,Klug CS





Has Abstract


2006-03-01 00:00:00














  • A new protocol for high-yield purification of recombinant human CXCL8((3-72))K11R/G31P expressed in Escherichia coli.

    abstract::The ELR-CXC chemokines are important to neutrophil inflammation in many acute and chronic diseases. Among them, CXCL8 (interleukin-8, IL-8), binds to both the CXCR1 and CXCR2 receptors with high affinity and the expression levels of CXCL8 are elevated in many inflammatory diseases. Recently, an analogue of human CXCL8...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Cheng HT,Huang KC,Yu HY,Gao KJ,Zhao X,Li F,Town J,Gordon JR,Cheng JW

    更新日期:2008-09-01 00:00:00

  • Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies.

    abstract::Antimicrobial peptide LL-37 plays an important role in human body's first line of defense against infection. To better understand the mechanism of action, it is critical to elucidate the three-dimensional structure of LL-37 in complex with bacterial membranes. We present a bacterial expression system that allows the i...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Li Y,Li X,Wang G

    更新日期:2006-06-01 00:00:00

  • A hydrophobic interaction chromatography strategy for purification of inactivated foot-and-mouth disease virus.

    abstract::A purification scheme based on hydrophobic interaction chromatography was developed to separate inactivated foot-and-mouth disease virus (FMDV) from crude supernatant. About 92% recovery and 8.8-fold purification were achieved on Butyl Sepharose 4 FF. Further purification on Superdex 200 resulted in another 29-fold pu...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Li H,Yang Y,Zhang Y,Zhang S,Zhao Q,Zhu Y,Zou X,Yu M,Ma G,Su Z

    更新日期:2015-09-01 00:00:00

  • Purification of proteins with native terminal sequences using a Ni(II)-cleavable C-terminal hexahistidine affinity tag.

    abstract::The role of the termini of protein sequences is often perturbed by remnant amino acids after the specific protease cleavage of the affinity tags and/or by the amino acids encoded by the plasmid at/around the restriction enzyme sites used to insert the genes. Here we describe a method for affinity purification of a met...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Abd Elhameed HAH,Hajdu B,Balogh RK,Hermann E,Hunyadi-Gulyás É,Gyurcsik B

    更新日期:2019-07-01 00:00:00

  • Production and characterization of biologically active human GM-CSF secreted by genetically modified plant cells.

    abstract::Human granulocyte-macrophage colony-stimulating factor (GM-CSF), a hemopoietic growth factor, was produced and secreted from tobacco cell suspensions. The GM-CSF cDNA was carried by a binary vector under the control of the CaMV 35S promoter and the T7 terminator. In addition, a 5'-nontranslated region from the tobacco...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: James EA,Wang C,Wang Z,Reeves R,Shin JH,Magnuson NS,Lee JM

    更新日期:2000-06-01 00:00:00

  • Construction, expression and purification of a novel CadF-based multiepitope antigen and its immunogenic polyclonal antibody specific to Campylobacter jejuni and Campylobacter coli.

    abstract::Campylobacteriosis is a disease in humans caused by the infection from Campylobacter spp. Human cases are mainly due to Campylobacter jejuni, although C. coli can cause gastroenteritis in humans as well. The bacteria are commensal in chicken tract and can be contaminated into chicken products during processing. Obviou...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Wenbap P,Seetang-Nun Y,Luangtongkum T,Khunrae P,Tuitemwong P,Rattanarojpong T

    更新日期:2021-04-01 00:00:00

  • Cloning, overexpression, folding and purification of a biosynthetically derived three disulfide scorpion toxin (BTK-2) from Mesobuthus tamulus.

    abstract::BTK-2, a 32 residue scorpion toxin initially identified in the venom of red Indian scorpion Mesobuthus tamulus was cloned, overexpressed and purified using Cytochrome b(5) fusion protein system developed in our laboratory. The synthetic gene coding for the peptide was designed taking into account optimal codon usage b...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Kumar GS,Sarma SP

    更新日期:2010-04-01 00:00:00

  • An improved method for high-level soluble expression and purification of recombinant amyloid-beta peptide for in vitro studies.

    abstract::Amyloid-beta (Aβ) peptide mediates several neurodegenerative diseases. The 42 amino acid (Aβ1-42) is the predominant form of peptide found in the neuritic plaques and has been demonstrated to be neurotoxic in vivo and in vitro. The availability of large quantities of Aβ peptide will help in several biochemical and bio...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Chhetri G,Pandey T,Chinta R,Kumar A,Tripathi T

    更新日期:2015-10-01 00:00:00

  • Expression, purification, and characterization of mouse glycine N-acyltransferase in Escherichia coli.

    abstract::Glycine N-acyltransferase (GLYAT) is a phase II metabolic detoxification enzyme for exogenous (xenobiotic) and endogenous carboxylic acids; consisting of fatty acids, benzoic acid, and salicylic acid. GLYAT catalyzes the formation of hippurate (N-benzoylglycine) from the corresponding glycine and benzoyl-CoA. Herein, ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Dempsey DR,Bond JD,Carpenter AM,Rodriguez Ospina S,Merkler DJ

    更新日期:2014-05-01 00:00:00

  • Large-scale preparation, purification, and crystallization of UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase from Escherichia coli.

    abstract::The UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli, an enzyme involved in the biosynthesis of the bacterial peptidoglycan monomer unit, was overproduced and purified to homogeneity on a large scale, yielding 4 mg of protein per liter of bacterial culture. Crystals of the complex with the subst...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Auger G,Martin L,Bertrand J,Ferrari P,Fanchon E,Vaganay S,Pétillot Y,van Heijenoort J,Blanot D,Dideberg O

    更新日期:1998-06-01 00:00:00

  • Efficient recovery of the functional IP10-scFv fusion protein from inclusion bodies with an on-column refolding system.

    abstract::A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpresse...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Guo JQ,Li QM,Zhou JY,Zhang GP,Yang YY,Xing GX,Zhao D,You SY,Zhang CY

    更新日期:2006-01-01 00:00:00

  • A new method for the purification of bioactive insulin-like growth factor-binding protein-3.

    abstract::We present a novel efficient procedure for high level purification of human IGFBP-3. Insulin-like growth factor-binding proteins (IGFBPs) are key regulators of insulin-like growth factor mediated signal transduction and thereby can profoundly influence cellular phenotypes. Certain IGFBPs, including IGFBP-3, have also ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Pircher H,Matscheski A,Laich A,Hermann M,Moser B,Viertler HP,Micutkova L,Lindner H,Sarg B,Zwerschke W,Jansen-Dürr P

    更新日期:2010-06-01 00:00:00

  • Expression of Sonic hedgehog-Fc fusion protein in Pichia pastoris. Identification and control of post-translational, chemical, and proteolytic modifications.

    abstract::We have investigated the suitability of Pichia pastoris as an expression system for the candidate therapeutic protein, Sonic hedgehog fused to an immunoglobulin Fc domain (Shh-Fc). Sonic hedgehog is a morphogen protein involved in the patterning of a wide range of tissues during animal embryogenesis. The presence of S...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Shapiro RI,Wen D,Levesque M,Hronowski X,Gill A,Garber EA,Galdes A,Strauch KL,Taylor FR

    更新日期:2003-06-01 00:00:00

  • A simplified method for purification of recombinant soluble DnaA proteins.

    abstract::An improved, simplified method for the purification of recombinant, tagged DnaA proteins is described. The presented protocol allowed us to purify soluble DnaA proteins from two different bacterial species: Helicobacter pylori and Streptomyces coelicolor, but it can most likely also be used for the isolation of DnaA p...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zawilak-Pawlik AM,Kois A,Zakrzewska-Czerwinska J

    更新日期:2006-07-01 00:00:00

  • Catalytic and cytotoxic activities of recombinant ricin A chain mutants with charged residues added at the carboxyl terminus.

    abstract::Ricin A chain (RTA) mutants which had been modified by the addition of three lysine residues, three lysines and an alanine, or six histidine residues at the carboxyl terminus were expressed in Escherichia coli. The recombinant proteins were purified to homogeneity by ion-exchange chromatography on CM-Sepharose CL-6B. ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Simpson JC,Roberts LM,Lord JM

    更新日期:1995-10-01 00:00:00

  • Bacterially expressed recombinant p68 RNA helicase is phosphorylated on serine, threonine, and tyrosine residues.

    abstract::We previously reported the expression and purification of recombinant p68 RNA helicase in a bacterial expression system. The recombinant p68 is an RNA-dependent ATPase and ATP-dependent RNA helicase. In the process of characterizing the ATPase and RNA unwinding activities of the recombinant p68, we observed that the b...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Yang L,Liu ZR

    更新日期:2004-06-01 00:00:00

  • Construction of high-density display of CD147 ectodomain on VCSM13 phage via gpVIII: effects of temperature, IPTG, and helper phage infection-period.

    abstract::Production of VCSM13 phage displaying a high density of CD147 ectodomain (CD147Ex) was achieved when culturing conditions were modulated. A phagemid expressing CD147Ex was constructed and used to produce phage display CD147Ex gpVIII fusion protein in TG1 Escherichia coli. Displaying of CD147Ex via gpVIII was successfu...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Intasai N,Arooncharus P,Kasinrerk W,Tayapiwatana C

    更新日期:2003-12-01 00:00:00

  • A-protein from achromogenic atypical Aeromonas salmonicida: molecular cloning, expression, purification, and characterization.

    abstract::Achromogenic atypical Aeromonas salmonicida is the causative agent of goldfish ulcer disease. Virulence of this bacterium is associated with the production of a paracrystalline outer membrane A-layer protein. The species-specific structural gene for the monomeric form of A-protein was cloned into a pET-3d plasmid in o...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Maurice S,Hädge D,Dekel M,Friedman A,Gertler A,Shoseyov O

    更新日期:1999-08-01 00:00:00

  • Baculovirus expression, purification, and characterization of human protein phosphatase 2A catalytic subunits alpha and beta.

    abstract::Protein phosphatase 2A (PP2A) contains a 36-kDa catalytic subunit (PP2Ac), a 65-kDa structural subunit (PR65/A), and a regulatory B subunit. The core enzyme consists of the structural and catalytic subunits. The catalytic subunit exists as two closely related isoforms, alpha and beta. Several natural toxins, including...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Ikehara T,Shinjo F,Ikehara S,Imamura S,Yasumoto T

    更新日期:2006-01-01 00:00:00

  • Optimization of human D-amino acid oxidase expression in Escherichia coli.

    abstract::Human D-amino acid oxidase (hDAAO) is a flavoprotein that plays a key role in the pathophysiology of schizophrenia. So far, the biochemical characterization of this enzyme has been hampered by the difficulty of expressing it in a common heterologous host such as Escherichia coli. Increasing amounts of recombinant hDAA...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Romano D,Molla G,Pollegioni L,Marinelli F

    更新日期:2009-11-01 00:00:00

  • Affinity purification of GST fusion proteins for immunohistochemical studies of gene expression.

    abstract::Fusion proteins expressed in bacteria are often insoluble or inefficiently purified by standard procedures previously reported to work well for the non-fused proteins. We report here a simple but general procedure that can be used to quickly customize and optimize the purification of milligram quantities of most GST f...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Mercado-Pimentel ME,Jordan NC,Aisemberg GO

    更新日期:2002-11-01 00:00:00

  • Overexpression and reconstitution of a Rieske iron-sulfur protein from the higher plant.

    abstract::The iron-sulfur protein subunit, known as the Rieske protein, is one of the central components of the cytochrome b(6)f complex residing in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overexpression in Escherichia coli of full-length and truncated Rieske (PetC) proteins from the...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Gubernator B,Seidler A,Rögner M,Szczepaniak A

    更新日期:2003-05-01 00:00:00

  • Transmembrane-sequence-dependent overexpression and secretion of glycoproteins in Saccharomyces cerevisiae.

    abstract::Protein expression using the secretory pathway in Saccharomyces cerevisiae can lead to high amounts of overexpressed and secreted proteins in culture supernatants in a short period of time. These post-translational modified expression products can be purified up to >90% in a single step. The overexpression and secreti...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Schuster M,Wasserbauer E,Aversa G,Jungbauer A

    更新日期:2001-02-01 00:00:00

  • Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions.

    abstract::Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Liu L,Spurrier J,Butt TR,Strickler JE

    更新日期:2008-11-01 00:00:00

  • Purification and characterization of a long-acting ciliary neurotrophic factor via genetically fused with an albumin-binding domain.

    abstract::Ciliary neurotrophic factor (CNTF) is a promising candidate for the treatment of neurodegenerative or metabolic diseases, but suffers rapid clearance in body. Herein we constructed a new long-acting recombinant human CNTF (rhCNTF) by genetic fusion with an albumin-binding domain (ABD) through a flexible peptide linker...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Xu L,Zhang C,Liu L,Zhang Y,Wang Q,Wang J,Liu Y,Su Z

    更新日期:2017-11-01 00:00:00

  • Expression, purification, crystallization, and preliminary X-ray analysis of the N-terminal domain of Escherichia coli adenylyl transferase.

    abstract::A soluble N-terminal domain of the Escherichia coli adenylyl transferase (ATase) is responsible for deadenylylation activity of the intact enzyme. Previous studies of the deadenylylation activity have involved a fragment, AT-N423 (residues 1 to 423), which was extended by 17 amino acids to give AT-N440. This new domai...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Xu Y,Wen D,Clancy P,Carr PD,Ollis DL,Vasudevan SG

    更新日期:2004-03-01 00:00:00

  • Amino-terminal charge affects the periplasmic accumulation of recombinant heregulin/EGF hybrids exported using the Escherichia coli alkaline phosphatase signal sequence.

    abstract::An Escherichia coli expression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA-hEGF junction influence the peripl...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Campion SR,Elsasser E,Chung R

    更新日期:1997-08-01 00:00:00

  • Extracellular production of active vibriolysin engineered by random mutagenesis in Escherichia coli.

    abstract::Vibriolysin, an extracellular protease of Vibrio proteolyticus, is synthesized as a preproenzyme. The N-terminal propeptide functions as an intramolecular chaperone and an inhibitor of the mature enzyme. Extracellular production of recombinant vibriolysin has been achieved in Bacillus subtilis, but not in Escherichia ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Sonoda H,Sugimura A

    更新日期:2008-12-01 00:00:00

  • High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts.

    abstract::(S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis catalyzes the formation of (S)-cyanohydrins from hydrocyanic acid and aldehydes or ketones. This enzyme accepts aliphatic, aromatic, and heterocyclic carbonyl compounds as substrates and is therefore considered a potent biocatalyst for the...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Hasslacher M,Schall M,Hayn M,Bona R,Rumbold K,Lückl J,Griengl H,Kohlwein SD,Schwab H

    更新日期:1997-10-01 00:00:00

  • Solution studies of recombinant human stromal-cell-derived factor-1.

    abstract::Stromal-cell-derived factor-1 (SDF-1alpha) is an 8-kDa chemokine that is constitutively expressed in bone-marrow-derived stromal cells and has been identified as a ligand for the CXCR4 receptor. We produced the chemokine recombinantly as methionine-SDF-1alpha in Escherichia coli without the leader peptide sequence. Th...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Holmes WD,Consler TG,Dallas WS,Rocque WJ,Willard DH

    更新日期:2001-04-01 00:00:00