Kinetic characterization and Mg2+ enhancement of Streptomyces griseocarneus sphingomyelinase C produced by recombinant Streptomyces lividans.


:Sphingomyelinase C (SMC) of the actinomycete, Streptomycesgriseocarneus NBRC13471, was constitutively expressed to high levels using Streptomyces lividans host and thereafter was extracellularly secreted into the cell culture. Purified SMC had a high specific activity (approximately 550-950 U/mg) and was obtained in high yields (approximately 120 mg/L of culture). SMC activity was enhanced by MgCl(2), and the maximum activity (542±25 U/mg) was observed in the presence of 1.5 mol/L (M) MgCl(2). Dynamic light scattering analysis proved that the highest specific SMC activity was obtained with the smallest mixed micelles of sphingomyelin (SM) and Triton X-100. The turnover rate (k(cat)), K(m) and k(cat)/K(m) values for SM were 346 s(-1), 0.458 mM, and 756 mM(-1)s(-1), respectively, in the presence of 1M MgCl(2). The k(cat) was strongly influenced by the MgCl(2) concentration. By contrast, the K(m) value was independent of the MgCl(2) concentration and was almost constant. Circular dichroism spectroscopy indicated that MgCl(2) did not cause local structural changes in SMC. From these results, we concluded that the SMC activity enhancement by MgCl(2) was caused by the increased specific surface area of the mixed micelles composed of substrate, SM, and Triton X-100.


Protein Expr Purif


Sugimori D,Matsumoto Y,Tomita Y,Murayama K,Ogino C




Has Abstract


2012-02-01 00:00:00














  • Expression of Pisum sativum SAD polypeptides in production hosts and in planta: tetrameric organization of the protein.

    abstract::In Pisum sativum, the short-chain alcohol dehydrogenase-like protein (SAD) gene family consists of at least three members (SAD-A, -B, and -C). Expression of two of these genes (SAD-A and -C) in Escherichia coli or Pichia pastoris resulted in full-length soluble proteins. Purified SAD-A was used as antigen for antibody...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Scherbak N,Brosché M,Ala-Häivälä A,Strid H,Ohrfelt A,Nilsson F,Strid A

    更新日期:2009-01-01 00:00:00

  • Hepatocyte growth factor (HGF) is a copper-binding protein: a facile probe for purification of HGF by immobilized Cu(II)-affinity chromatography.

    abstract::Hepatocyte growth factor (HGF) is a multifunctional protein expressed in a variety of cell types and tissues. Here we describe a novel one-step method to separate and identify HGF, based on a unique interaction between HGF and Cu(II). Conditioned medium (CM) from mouse 3T3-L1 adipocytes which contains HGF or purified ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Rahimi N,Etchells S,Elliott B

    更新日期:1996-05-01 00:00:00

  • High-level synthesis of recombinant murine endostatin in Chinese hamster ovary cells.

    abstract::Endostatin, a carboxy-terminal fragment of collagen XVIII, has been shown to act as an anti-angiogenic agent that specifically inhibits proliferation of endothelial cells and growth of various primary tumors. Here, we describe the expression by Chinese hamster ovary (CHO) cells of murine endostatin and of a tagged-fus...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Chura-Chambi RM,Tornieri PH,Spencer PJ,Nascimento PA,Mathor MB,Morganti L

    更新日期:2004-05-01 00:00:00

  • Expression and purification of functional human glycogen synthase-1 (hGYS1) in insect cells.

    abstract::We have successfully expressed and purified active human glycogen synthase-1 (hGYS1). Successful production of the recombinant hGYS1 protein was achieved by co-expression of hGYS1 and rabbit glycogenin (rGYG1) using the MultiBac baculovirus expression system (BEVS). Functional measurements of activity ratios of hGYS1 ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Khanna M,Imasaki T,Chikwana VM,Perez-Miller S,Hunter GO,Mosley A,Takagi Y,Hurley TD

    更新日期:2013-08-01 00:00:00

  • Functional expression, purification, and characterization of human Flt3 ligand in the Pichia pastoris system.

    abstract::Flt3 ligand (FL) is a potent hematopoietic cytokine that affects the growth and differentiation of hematopoietic progenitor and stem cells both in vivo and in vitro. Pichia pastoris transformants secreting high-level rhFL were obtained using 'yeastern blotting' method and the expression level in liquid was about 30 mg...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zhang YL,Chen SS,Yang KG,Su L,Deng YC,Liu CZ

    更新日期:2005-08-01 00:00:00

  • Development of a bacterial system for high yield expression of fully functional adrenal cytochrome b561.

    abstract::Adrenal cytochrome b561 (cyt b561) is the prototypical member of an emerging family of proteins that are distributed widely in vertebrate, invertebrate and plant tissues. The adrenal cytochrome is an integral membrane protein with two b-type hemes and six predicted transmembrane helices. Adrenal cyt b561 is involved i...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Liu W,Rogge CE,Kamensky Y,Tsai AL,Kulmacz RJ

    更新日期:2007-12-01 00:00:00

  • Expression of soluble and active interferon consensus in SUMO fusion expression system in E. coli.

    abstract::Protein production can be improved if methods for soluble protein expression are developed. Interferon consensus (IFN-con) is used to treat hepatitis C. IFN-con has superior activity compared to other clinically used interferon α subtypes. However IFN-con is a challenging protein to produce in a soluble form using an ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Peciak K,Tommasi R,Choi JW,Brocchini S,Laurine E

    更新日期:2014-07-01 00:00:00

  • High yield expression and purification of isotopically labelled human endothelin-1 for use in NMR studies.

    abstract::Human endothelin-1 (ET-1) is a potent vasocontractile 21-residue peptide hormone with significant pharmacological importance. An efficient and straightforward expression strategy that enables cost-effective incorporation of stable isotopes is not available thus far. In this report, we describe a cost-effective express...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Mac TT,Beyermann M,Pires JR,Schmieder P,Oschkinat H

    更新日期:2006-08-01 00:00:00

  • A single-step purification of biologically active recombinant human interleukin-5 from a baculovirus expression system.

    abstract::Recombinant human interleukin-5 (rhIL-5) was expressed in baculovirus-infected insect cells and purified to homogeneity from the culture medium in a single chromatographic step. Beginning with a cDNA encoding the full-length precursor form of human IL-5, including the authentic secretory leader sequence, recombinant b...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Brown PM,Scheid MP,O'Neill GP,Tagari PC,Nicholson DW

    更新日期:1995-02-01 00:00:00

  • Gene cloning, bacterial expression, in vitro refolding, and characterization of a single-chain Fv antibody against PreS1(21-47) fragment of HBsAg.

    abstract::The murine monoclonal antibody 125E11 is an IgG which recognizes PreS1(21-47) fragment of large hepatitis B surface antigen. It has been successfully used for clinical detection of HBV virion in serum of hepatitis B patients. In present study, the genes of variable region in heavy chain (VH) and light chain (VL) of 12...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Yang X,Hu W,Li F,Xia H,Zhang Z

    更新日期:2005-06-01 00:00:00

  • Purification and characterization of recombinant rabbit cytosolic serine hydroxymethyltransferase.

    abstract::A rabbit liver cDNA library in phage lambdagt10 was screened using the coding cDNA for human cytosolic serine hydroxymethyltransferase. A clone of 1754 bp was isolated and the nucleotide sequence showed an open reading frame of 1455 bp, which coded for rabbit cytosolic serine hydroxymethyltransferase and was flanked b...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: di Salvo ML,Delle Fratte S,De Biase D,Bossa F,Schirch V

    更新日期:1998-07-01 00:00:00

  • High yield expression, refolding, and characterization of recombinant interferon alpha2/alpha8 hybrids in Escherichia coli.

    abstract::Interferons (IFNs) are a family of pleiotropic cytokines used for the treatment of various viral infections and cancers. The low-cost production of IFNs with high biological value and the discovery of IFNs with improved properties are important for the treatment of these diseases as well as for understanding the physi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Platis D,Foster GR

    更新日期:2003-10-01 00:00:00

  • A three-step purification of manganese superoxide dismutase from human liver on both large and small scales.

    abstract::A new method for the purification of manganese superoxide dismutase from human liver is described. The procedure involves essentially three steps: DEAE-cellulose, hydroxylapatite, and butyl-Toyopearl chromatographies. The method has several advantages: (i) its simplicity and rapidity (it takes less than 3 days), (ii) ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Matsuda Y,Suzuki K,Ookawara T,Nakata T,Seo HG,Kawata S,Tarui S,Deutsch HF,Taniguchi N

    更新日期:1991-04-01 00:00:00

  • Expression, purification, and initial structural characterization of nonstructural protein 2B, an integral membrane protein of Dengue-2 virus, in detergent micelles.

    abstract::Dengue virus causes serious diseases affecting people in tropical and sub-tropical regions. The nonstructural (NS) protein 2B is an integral membrane protein and important for the regulation of viral protease NS3, which is significant for virus replication. The NS2B-NS3 complex is an important drug target for treating...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Huang Q,Chen AS,Li Q,Kang C

    更新日期:2011-12-01 00:00:00

  • Thermostable tyrosine phenol-lyase of Symbiobacterium sp. SC-1: gene cloning, sequence determination, and overproduction in Escherichia coli.

    abstract::During the screening for tyrosine phenol-lyase-producing thermophiles, we isolated an obligatory symbiotic thermophile, Symbiobacterium sp. SC-1, which grew only in coculture with Bacillus sp. SK-1. A gene encoding thermostable tyrosine phenol-lyase (TPL) was cloned from the genomic DNA of the Symbiobacterium sp. SC-1...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Lee SG,Hong SP,Choi YH,Chung YJ,Sung MH

    更新日期:1997-12-01 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

  • Expression of a soluble and activatable form of bovine procarboxypeptidase A in Escherichia coli.

    abstract::Bovine pancreatic procarboxypeptidase A has been overexpressed in a soluble and activatable form in Escherichia coli. When the protein was expressed under the control of bacteriophage T7 promoter in E. coli ADA494 (a thioredoxin reductase deficient bacteria), a thioredoxin fusion protein was produced at relatively hig...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Seddi R,Chaix JC,Puigserver A,Guo XJ

    更新日期:2003-02-01 00:00:00

  • Expression and structural characterization of human translocase of inner membrane of mitochondria Tim50.

    abstract::The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim50 is a major receptor in TIM23 complex, which spans the inner membrane with a single transmembrane segment and exposes a large hydrophilic domain in the interme...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zhang Y,Xu Y,Zhao Q,Ji Z,Li Q,Li SJ

    更新日期:2011-11-01 00:00:00

  • Expression and purification of human antimicrobial peptide, dermcidin, in Escherichia coli.

    abstract::Human dermcidin, an anionic antimicrobial peptide expressed in the pons of the brain and the sweat glands, displays antimicrobial activity against pathogenic microorganisms such as Staphylococcus aureus and Candida albicans. Here, we describe the recombinant production of a 48 amino acid dermcidin variant with C-termi...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Cipáková I,Gasperík J,Hostinová E

    更新日期:2006-02-01 00:00:00

  • Binding studies using Pichia pastoris expressed human aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins.

    abstract::The aryl hydrocarbon receptor (AHR) is a transcription factor which activates gene transcription by binding to its corresponding enhancer as the heterodimer, which is consisted of AHR and the aryl hydrocarbon receptor nuclear translocator (ARNT). Human AHR can be rather difficult to study, when compared among the AHR ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zheng Y,Xie J,Huang X,Dong J,Park MS,Chan WK

    更新日期:2016-06-01 00:00:00

  • Improved performance of column chromatography by arginine: dye-affinity chromatography.

    abstract::Arginine has been effectively used in various column chromatographies for improving recovery and resolution, and suppressing aggregation. Here, we have tested the effectiveness of arginine as an eluent in dye-affinity column chromatography using Blue-Sepharose, which binds enzymes requiring adenyl-containing cofactors...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Arakawa T,Ejima D,Tsumoto K,Ishibashi M,Tokunaga M

    更新日期:2007-04-01 00:00:00

  • Dog zona pellucida glycoprotein-3 (ZP3): expression in Escherichia coli and immunological characterization.

    abstract::An internal fragment (978 bp) corresponding to the dog zona pellucida glycoprotein-3 (DZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was amplified by polymerase chain reaction from a full-length cDNA clone. The amplified SacI and PstI restricted fragment was cloned in-fra...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Santhanam R,Panda AK,Kumar VS,Gupta SK

    更新日期:1998-04-01 00:00:00

  • Functional recombinant rabbit muscle phosphoglucomutase from Escherichia coli.

    abstract::The gene coding for phosphoglucomutase (PGM) from Oryctolagus cuniculus (rabbit) has been expressed in Escherichia coli under a T7 expression system with a His-tag. About half of the expressed PGM protein was present in inclusion bodies, but this protein was inactive when solubilized. The protein in the soluble cell f...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Chae YK,Markley JL

    更新日期:2000-10-01 00:00:00

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

    abstract::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...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Chu X,Yu W,Chen G,Li D

    更新日期:2003-10-01 00:00:00

  • Rapid generation of stable cell lines expressing corticotropin-releasing hormone receptor for drug discovery.

    abstract::Human HEK293 cells that stably express the Epstein Barr nuclear antigen 1 (EBNA1) support the episomal replication of plasmids containing the Epstein Barr virus origin of replication (EBV oriP). A 293EBNA (293E) cell line expressing the human corticotropin-releasing hormone receptor subtype I (CRHR1) from an episomal ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Horlick RA,Sperle K,Breth LA,Reid CC,Shen ES,Robbins AK,Cooke GM,Largent BL

    更新日期:1997-04-01 00:00:00

  • Preparation of soluble isotopically labeled NKp30, a human natural cytotoxicity receptor, for structural studies using NMR.

    abstract::Using a codon-optimized gene fragment, we report remarkable yields for extracellular domain of human NK cell receptor (NKp30ex) when produced on M9 minimal medium, even with low (2g/L) glucose concentration. The yields were identical using media containing (15)NH(4)Cl or (15)NH(4)Cl in combination with all-(13)C-d-glu...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Grave L,Tůmová L,Mrázek H,Kavan D,Chmelík J,Vaněk O,Novák P,Bezouška K

    更新日期:2012-12-01 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

  • Expression, purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli.

    abstract::Enteropeptidase (synonym:enterokinase, EC is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Gasparian ME,Ostapchenko VG,Schulga AA,Dolgikh DA,Kirpichnikov MP

    更新日期:2003-09-01 00:00:00

  • Cloning, expression, identification and characterization of borneol dehydrogenase isozymes in Pseudomonas sp. TCU-HL1.

    abstract::Borneol is a bicyclic plant monoterpene. It can be degraded by soil microorganisms through the conversion of borneol dehydrogenase (BDH) and a known camphor degradation pathway. Recombinant BDH from Pseudomonas sp. TCU-HL1 was produced in the form of inclusion body. The refolded BDH1 tends to precipitate. Insoluble re...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Khine AA,Lu PC,Ko TP,Huang KF,Chen HP

    更新日期:2020-11-01 00:00:00

  • Expression, purification, and antifreeze activity of carrot antifreeze protein and its mutants.

    abstract::Antifreeze proteins (AFPs) enable organisms to survive under freezing or sub-freezing conditions. AFPs have a great potential in the low temperature storage of cells, tissues, organs, and foods. This process will require a large number of recombinant AFPs. In the present study, the recombinant carrot AFP was highly ex...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zhang DQ,Liu B,Feng DR,He YM,Wang JF

    更新日期:2004-06-01 00:00:00