Expression, purification, and characterization of His-tagged human mitochondrial 2,4-dienoyl-CoA reductase.


:Mitochondrial 2,4-dienoyl-CoA reductase is a key enzyme for the beta-oxidation of unsaturated fatty acids. The cDNA of the full-length human mitochondrial 2,4-dienoyl-CoA reductase was previously cloned as pUC18::DECR. PCR methodologies were used to subclone the genes encoding various truncated human mitochondrial 2,4-dienoyl-CoA reductases from pUC18::DECR with primers that were designed to add six continuous histidine codons to the 3' or 5' primer. The PCR products were inserted into pLM1 expression vectors and overexpressed in Escherichia coli. A highly active truncated soluble protein was expressed and purified with a nickel HiTrap chelating metal affinity column to apparent homogeneity based on Coomassie blue-stained SDS-PAGE. The molecular weight of the protein subunit was 34 kDa. The purified protein is highly stable at room temperature, which makes it potentially valuable for protein crystallization. KM of 26.5 +/- 3.8 microM for 2,4-hexadienoyl-CoA, KM of 6.22 +/- 2.0 microM for 2,4-decadienoyl-CoA, and KM of 60.5 +/- 19.7 microM for NADPH, as well as Vmax of 7.78 +/- 1.08 micromol/min/mg for 2,4-hexadienoyl-CoA and Vmax of 0.74 +/- 0.07 micromol/min/mg for 2,4-decadienoyl-CoA were determined on kinetic study of the purified protein. The one-step purification of the highly active human mitochondrial 2,4-dienoyl-CoA reductase will greatly facilitate further investigation of this enzyme through site-directed mutagenesis and enzyme catalyzed reactions with substrate analogs as well as protein crystallization for solving its three-dimensional structure.


Protein Expr Purif


Chu X,Yu W,Chen G,Li D





Has Abstract


2003-10-01 00:00:00














  • Zebrafish Sp1-like protein is structurally and functionally comparable to human Sp1.

    abstract::The transcription factor Sp1 is a regulator of TATA-less genes. It belongs to the Cys₂-His₂ zinc finger domain-containing family. A zebrafish cDNA encoding a peptide homologous to mammalian Sp1 was cloned and inserted into a pET43.1a vector and expressed in Escherichia coli Rosetta (DE3) cells as a Nus-His-tag fusion ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Lin CJ,Hsiao TH,Chung YS,Chang WN,Yeh TM,Chen BH,Fu TF

    更新日期:2011-03-01 00:00:00

  • Purified recombinant Escherichia coli ketol-acid reductoisomerase is unsuitable for use in a coupled assay of acetohydroxyacid synthase activity due to an unexpected side reaction.

    abstract::Ketol-acid reductoisomerase (EC catalyzes the conversion of 2-aceto-2-hydroxyacids to 2-keto-3-hydroxyacids and their subsequent reduction by NADPH to 2,3-dihydroxyacids. The gene encoding the Escherichia coli enzyme was cloned and expressed as a hexahistidine-tagged fusion protein and the recombinant enzyme...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Hill CM,Duggleby RG

    更新日期:1999-02-01 00:00:00

  • Addressing Shewanella oneidensis "cytochromome": the first step towards high-throughput expression of cytochromes c.

    abstract::Integrated studies that address proteins structure and function in the new era of systems biology and genomics often require the application of high-throughput approaches for parallel production of many different purified proteins from the same organism. Cytochromes c-electron transfer proteins carrying one or more he...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Londer YY,Giuliani SE,Peppler T,Collart FR

    更新日期:2008-11-01 00:00:00

  • Efficient expression of SRK intracellular domain by a modeling-based protein engineering.

    abstract::S-locus protein kinase (SRK) is a receptor kinase that plays a critical role in self-recognition in the Brassicaceae self-incompatibility (SI) response. SRK is activated by binding of its ligand S-locus protein 11 (SP11) and subsequently induced phosphorylation of the intracellular kinase domain. However, a detailed a...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Murase K,Hirano Y,Takayama S,Hakoshima T

    更新日期:2017-03-01 00:00:00

  • Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein-chitin-binding domain (CBD) system.

    abstract::The Staphylococcus simulans gene encoding lysostaphin has been PCR amplified from pRG5 recombinant plasmid (ATCC 67076) and cloned into Escherichia coli expression pTYB12 vector (IMPACT-CN System, New England BioLabs) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag. The...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Szweda P,Pladzyk R,Kotlowski R,Kur J

    更新日期:2001-08-01 00:00:00

  • Recombinant expression of biologically active rat leptin in Escherichia coli.

    abstract::Leptin is a 16-kDa nonglycosylated hormone that is produced in mature adipocytes and which acts primarily in the hypothalamus to reduce food intake and body weight. While the rat is a representative laboratory animal model in obesity research, so far recombinant rat leptin was not available. In the present study, rat ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Park JH,Lee HH,Na SY,Ju SK,Lee YJ,Lee MK,Kim KL

    更新日期:2001-06-01 00:00:00

  • Cloning, expression, purification, and characterization of a glutamate-specific endopeptidase from Bacillus licheniformis.

    abstract::A gene encoding a glutamate-specific endopeptidase (GSE) from Bacillus licheniformis (BL) has been cloned in Escherichia coli cells. The recombinant protein was expressed as cytoplasmic insoluble inclusion bodies. Immobilized metal affinity chromatography was employed to purify the protein, and then a 27-kDa GSE inter...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Ye W,Liu J,Wang H,Wang J,Wang X

    更新日期:2012-03-01 00:00:00

  • Immediate-early baculovirus vectors for foreign gene expression in transformed or infected insect cells.

    abstract::Baculovirus expression vectors are used routinely for foreign gene expression and are under intense development as improved biological pesticides. Conventional baculovirus expression vectors are recombinant viruses that can express a foreign gene in insect cells under the control of the polyhedrin promoter, which prov...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Jarvis DL,Weinkauf C,Guarino LA

    更新日期:1996-09-01 00:00:00

  • Purification and characterization of fibrinolytic enzyme from cultured mycelia of Armillaria mellea.

    abstract::A fibrinolytic enzyme was purified from the cultured mycelia of Armillaria mellea by ion-exchange chromatography followed by gel filtration, and was designated A. mellea metalloprotease (AMMP). The purification protocol resulted in a 627-fold purification of the enzyme, with a final yield of 6.05%. The apparent molecu...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Lee SY,Kim JS,Kim JE,Sapkota K,Shen MH,Kim S,Chun HS,Yoo JC,Choi HS,Kim MK,Kim SJ

    更新日期:2005-09-01 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

  • Bacillus subtilis alkaline phosphatase IV acquires activity only late at the stationary phase when produced in Escherichia coli. Overexpression and characterization of the recombinant enzyme.

    abstract::The availability of recombinant monomeric alkaline phosphatase (AP) is highly desirable in analytical applications involving AP fusion proteins. The cobalt-dependant alkaline phosphatase IV from Bacillus subtilis (BSAP), which was reported to be strongly monomeric, was overexpressed in Escherichia coli using pET autoi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Koksharov M,Lv C,Zhai X,Ugarova N,Huang E

    更新日期:2013-08-01 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

  • A simple and efficient method for generating high-quality recombinant Mical enzyme for in vitro assays.

    abstract::We have recently identified a new family of multidomain oxidoreductase (redox) enzymes, the MICALs, that directly regulate the actin cytoskeletal elements necessary for the morphology, motility, and trajectory of cells. Our genetic assays reveal that Mical is both necessary and sufficient for actin organization and ce...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Wu H,Hung RJ,Terman JR

    更新日期:2016-11-01 00:00:00

  • Improved soluble expression and characterization of the Hc domain of Clostridium botulinum neurotoxin serotype A in Escherichia coli by using a PCR-synthesized gene and a Trx co-expression strain.

    abstract::Botulinum neurotoxin serotype A (BoNT/A) is an extremely potent bacterial protein toxin. The Hc fragment of BoNT/A (AHc) was shown to be non-toxic, antigenic, and capable of eliciting a protective immunity in animals challenged with homologous BoNT. In this study, we synthesized AHc gene by using T4 DNA ligase and PCR...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Chen R,Shi J,Cai K,Tu W,Hou X,Liu H,Xiao L,Wang Q,Tang Y,Wang H

    更新日期:2010-05-01 00:00:00

  • Novel thermostable ssDNA-binding proteins from Thermus thermophilus and T. aquaticus-expression and purification.

    abstract::Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination, and repair in bacteria, archaea, and eukarya. We report here the identification, expression, and purification of the SSB-like proteins of the thermophilic bacteria Thermus thermophilus and T. aquaticus. The nucleotide (...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Dabrowski S,Olszewski M,Piatek R,Kur J

    更新日期:2002-10-01 00:00:00

  • Heterologous expression and in vitro assembly of the transmembrane cytochrome b6.

    abstract::Folding and assembly studies with alpha-helical membrane proteins are often hampered by the absence of high-level expression systems as well as by missing suitable in vitro refolding procedures. Experimental constraints and requirements for heterologous expression and in vitro assembly of cytochrome b6 have been exami...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Prodöhl A,Dreher C,Hielscher R,Hellwig P,Schneider D

    更新日期:2007-12-01 00:00:00

  • Recombinant production of a VL single domain antibody in Escherichia coli and analysis of its interaction with peptostreptococcal protein L.

    abstract::A kappa-light chain from a Fab expression system was truncated by the insertion of a stop codon in the gene sequence to produce a variable light (VL) single domain antibody (dAb). Here, we describe the expression of dAb in the periplasm of Escherichia coli through fermentation in a defined media. Immunoglobulin bindin...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Cossins AJ,Harrison S,Popplewell AG,Gore MG

    更新日期:2007-02-01 00:00:00

  • Preventing protein aggregation by its hyper-acidic fusion cognates in Escherichia coli.

    abstract::Preventing protein aggregation is crucial for various protein studies, and has a large potential for remedy of protein misfolding or aggregates-linked diseases. In this study, we demonstrated the hyper-acidic protein fusion partners, which were previously reported to enhance the soluble expression of aggregation-prone...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zou Z,Fan Y,Zhang C

    更新日期:2011-11-01 00:00:00

  • Secretory expression and purification of Aspergillus niger glucose oxidase in Saccharomyces cerevisiae mutant deficient in PMR1 gene.

    abstract::The gene encoding glucose oxidase (GOD) from Aspergillus niger was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Six consecutive histidine residues were fused to the C-terminus of GOD to facilitate purification. The recombinant GOD-His(6) secreted by S. cerevisiae migrated as a broad diffuse ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Ko JH,Hahm MS,Kang HA,Nam SW,Chung BH

    更新日期:2002-08-01 00:00:00

  • High-level expression and purification of a recombinant hBD-1 fused to LMM protein in Escherichia coli.

    abstract::In this work, we present the production of an active 43 aa recombinant human beta-defensin-1 (rhBD-1(43)) in Escherichia coli AD202 cells using specific pLMM1-rhBD-1 expression system. Unique solubility properties of the C-terminal fragment of light meromyosin (LMM) allowed us to overcome foreseeable problems with iso...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Cipáková I,Hostinová E,Gasperík J,Velebný V

    更新日期:2004-09-01 00:00:00

  • Generation and expression of a minimal hybrid Ig-receptor formed between single domains from proteins L and G.

    abstract::The Ig-binding properties of protein L from Peptostreptococcus magnus and protein G from Streptococcus have been successfully combined through the construction of a novel hybrid protein, consisting of a single Ig-binding domain from each protein. The biophysical and biochemical properties of this construct have been c...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Harrison SL,Housden NG,Bottomley SP,Cossins AJ,Gore MG

    更新日期:2008-03-01 00:00:00

  • Heterologous expression and pro-peptide supported refolding of the high specific endopeptidase Lys-C.

    abstract::The high specific lysyl endopeptidase (Lys-C; EC is often used for the initial fragmentation of polypeptide chains during protein sequence analysis. However, due to its specificity it could be a useful tool for the production of tailor-made protein hydrolysates with for example bioactive or techno functiona...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Stressler T,Eisele T,Meyer S,Wangler J,Hug T,Lutz-Wahl S,Fischer L

    更新日期:2016-02-01 00:00:00

  • Increasing the expression levels of papillomavirus major capsid protein in Escherichia coli by N-terminal deletion.

    abstract::The major capsid protein L1 of human papillomavirus (HPV) contains the immunodominant neutralization epitopes of the virus and can auto-assembles to form virus-like particles (VLPs). Therefore, HPV L1 capsid proteins have been well investigated as potential vaccine candidates. To express large quantities of human papi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Ma Z,Chen B,Zhang F,Yu M,Liu T,Liu L

    更新日期:2007-11-01 00:00:00

  • High-level extracellular production of Rhizopus oryzae lipase in Pichia pastoris via a strategy combining optimization of gene-copy number with co-expression of ERAD-related proteins.

    abstract::Rhizopus oryzae lipase (ROL) is an important industrial enzyme limited in application due to its low production in native strains. Here, we used a new combined strategy to overexpress ROL in Pichia pastoris. An efficient method based on bio-brick was developed to construct a series of vectors harboring different copy ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Jiao L,Zhou Q,Su Z,Xu L,Yan Y

    更新日期:2018-07-01 00:00:00

  • Expression and refolding of bioactive α-bungarotoxin V31 in E. coli.

    abstract::In order to obtain bioactive α-bungarotoxin (αBtx) using recombinant protein technique, a codon-optimized synthetic gene was expressed in fusion with the N-terminal 10-His-tag and C-terminal Strep-tag in Escherichia coli. Further optimization through site-directed mutagenesis enabled moderate expression of the protein...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Xu J,Li J,Wu X,Song C,Lin Y,Shen Y,Ye W,Sun C,Wang X,Li Z,Liu Y,Wei L,Li Z,Xu Z

    更新日期:2015-06-01 00:00:00

  • Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4.

    abstract::Cytosine-specific DNA methyltransferases are important enzymes in most living organisms. In prokaryotes, most DNA methyltransferases are members of the type II restriction-modification system where they methylate host DNA, thereby protecting it from digestion by the accompanying restriction endonucleases. DNA methyltr...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Grgic M,Williamson A,Kjæreng Bjerga GE,Altermark B,Leiros I

    更新日期:2018-10-01 00:00:00

  • High-level expression and purification of active scorpion long-chain neurotoxin BjαIT from Pichia pastoris.

    abstract::As an insect-selective neurotoxin, scorpion long-chain BjαIT is a promising prospect for insecticidal application; however, the difficulty of obtaining natural BjαIT represents the major obstacle preventing analysis of its insecticidal activity against agricultural insect pests. Here, we screened recombinant Pichia pa...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Li H,Xia Y

    更新日期:2018-12-01 00:00:00

  • Baculovirus expression and purification of a soluble, mutant G-protein alpha subunit.

    abstract::The cDNA for the alpha i1 protein that had undergone site-directed mutagenesis to change glycine-2 to alanine was ligated into a baculovirus transfer vector. A recombinant virus was obtained by transfecting Sf9 cells with both the wild-type baculovirus DNA and the transfer vector and screening for recombinant plaques....

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Jones TL,Woodard C,Spiegel AM

    更新日期:1993-02-01 00:00:00

  • Antigen-binding properties of monoclonal antibodies reactive with human TATA-binding protein and use in immunoaffinity chromatography.

    abstract::The TATA-binding protein (TBP) plays a central role in the assembly of most eukaryotic transcription initiation complexes. We have characterized 3 monoclonal antibodies (mAbs) that react in the far amino-terminal (N-terminal) domain of the human TBP molecule (residues 1-99). One of these mAbs (designated 1TBP22) is a ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Thompson NE,Foley KM,Burgess RR

    更新日期:2004-08-01 00:00:00

  • Heterologous production, purification and characterization of enzymatically active Sindbis virus nonstructural protein nsP1.

    abstract::Alphavirus nonstructural protein nsP1 possesses distinct methyltransferase (MTase) and guanylyltransferase (GTase) activities involved in the capping of viral RNAs. In alphaviruses, the methylation of GTP occurs before RNA transguanylation and nsP1 forms a covalent complex with m(7)GMP unlike the host mRNA guanylyltra...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Tomar S,Narwal M,Harms E,Smith JL,Kuhn RJ

    更新日期:2011-10-01 00:00:00