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


: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 and the nucleotide sequence of the TPL structural gene was determined. The gene consists of 1374 base pairs encoding a polypeptide of 458 amino acid residues; the molecular mass of the enzyme subunit is estimated to be 52,196 Da. The structural gene of TPL was amplified by PCR, blunt-ended, and ligated into the NcoI-HindIII site of plasmid pTrc99A to construct an expression vector for the overproduction of the thermostable TPL. The level of thermostable TPL production was about 15% of the total soluble proteins of Escherichia coli extract. The enzyme was purified to homogeneity from the E. coli extract with an overall yield of 48%.


Protein Expr Purif


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




Has Abstract


1997-12-01 00:00:00














  • Large-scale expression and purification of a soluble form of the pleckstrin homology domain of the human protooncogenic serine/threonine protein kinase PKB (c-akt) in Escherichia coli.

    abstract::The protooncogenic serine/threonine protein kinase PKB contains an amino-terminal pleckstrin homology (PH) domain which binds phosphatidylinositides. The PH domain, composed of approximately 100 loosely conserved amino acids, is found in many proteins, including kinases, phospholipases C, GTPases, GTPase-activating pr...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Ingley E,Hemmings BA

    更新日期:1999-11-01 00:00:00

  • Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization.

    abstract::Mammalian α-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of α-defensins, large amounts of α-defensins are essential. Although many expression systems for the production of recombinant α-defensins have been developed,...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Tomisawa S,Sato Y,Kamiya M,Kumaki Y,Kikukawa T,Kawano K,Demura M,Nakamura K,Ayabe T,Aizawa T

    更新日期:2015-08-01 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

  • Expression of non-toxic mutant of Escherichia coli heat-labile enterotoxin in tobacco chloroplasts.

    abstract::Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-la...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Kang TJ,Han SC,Kim MY,Kim YS,Yang MS

    更新日期:2004-11-01 00:00:00

  • Expression, purification, and characterization of deglycosylated human pro-prostate-specific antigen.

    abstract::Wild-type and deglycosylated forms of human prostate-specific antigen were expressed in Chinese hamster ovary (CHO) cells as zymogens. ProPSA was collected from conditioned medium and purified using a single cation-exchange chromatographic step for the deglycosylated form and cation-exchange followed by gel filtration...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Bowman KK,Clark J,Yu L,Mortara K,Radika K,Wang J,Zhan H

    更新日期:2000-12-01 00:00:00

  • An anti-peptide monoclonal antibody recognizing the tobacco etch virus protease-cleavage sequence and its application to a tandem tagging system.

    abstract::Peptide-based affinity tags are commonly used in recombinant production/purification of proteins, and are often preceded or followed by a protease recognition sequence to allow tag removal. We describe a rat monoclonal antibody 2H5 recognizing an undecapeptide tag called "eTev", which contains a recognition sequence f...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Tabata S,Kitago Y,Fujii Y,Mihara E,Tamura-Kawakami K,Norioka N,Takahashi K,Kaneko MK,Kato Y,Takagi J

    更新日期:2018-07-01 00:00:00

  • Construction of an expression and site-directed mutagenesis system of haloalkane dehalogenase in Escherichia coli.

    abstract::Haloalkane dehalogenase from Xanthobacter autotrophicus was efficiently expressed in Escherichia coli BL21 (DE3) and E. coli JM101. After introduction of restriction sites by PCR the haloalkane dehalogenase gene (dhlA) was translationally fused behind the T7 (phi 10), trc, and tac promoters. This resulted in expressio...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Schanstra JP,Rink R,Pries F,Janssen DB

    更新日期:1993-10-01 00:00:00

  • Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells.

    abstract::Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni(2+)-NTA, and POROS Q columns obtained approximately 100mg of protein...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Emmons TL,Mathis KJ,Shuck ME,Reitz BA,Curran DF,Walker MC,Leone JW,Day JE,Bienkowski MJ,Fischer HD,Tomasselli AG

    更新日期:2009-06-01 00:00:00

  • Expression in Escherichia coli of the elongation factor 1beta gene and its nucleotide T160C mutant from the archaeon Sulfolobus solfataricus.

    abstract::The guanine nucleotide exchange factor EF-1beta gene from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-1beta) was amplified by PCR and cloned into the pT7-7 expression vector. One of four selected clones harbored the T160C nucleotide substitution leading to the Y54H amino acid change in a hydrophobic r...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Ianniciello G,Masullo M,Raimo G,Arcari P,Bocchini V

    更新日期:1998-02-01 00:00:00

  • Bacterial expression and antibiotic activities of recombinant variants of human β-defensins on pathogenic bacteria and M. tuberculosis.

    abstract::Five variants of human β-defensins (HBDs) were expressed in Escherichia coli using two vector systems (pET28a(+) and pQE30) with inducible expression by IPTG. The last vector has not been previously reported as an expression system for HBDs. The recombinant peptides were different in their lengths and overall charge. ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Corrales-Garcia L,Ortiz E,Castañeda-Delgado J,Rivas-Santiago B,Corzo G

    更新日期:2013-05-01 00:00:00

  • Expressed protein ligation using an N-terminal cysteine containing fragment generated in vivo from a pelB fusion protein.

    abstract::Advances in expressed protein ligation (EPL) methods that permit specific introduction of unique modifications into proteins have facilitated protein engineering, structure-function and protein interaction studies. An EPL-generated hybrid exchangeable apolipoprotein has been constructed from recombinant fragments of a...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Hauser PS,Ryan RO

    更新日期:2007-08-01 00:00:00

  • Heterologous protein production in Escherichia coli using the propionate-inducible pPro system by conventional and auto-induction methods.

    abstract::We examined expression of two plant genes encoding coclaurine N-methyltransferase (CMT) and norcoclaurine synthase (NCS) in Escherichia coli from the Salmonella entericaprpBCDE promoter (P(prpB)) and compared it to that from the strongest IPTG-inducible promoter, P(T7). In contrast to our previous study showing slight...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Lee SK,Keasling JD

    更新日期:2008-10-01 00:00:00

  • A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli.

    abstract::To facilitate studies of multicomponent protein complexes, I have developed an Escherichia coli expression system which coexpresses up to four polypeptides from a single plasmid. The modular nature of the system enables efficient subcloning of a gene into each of the 4 cassettes in the polycistronic expression vector....

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Tan S

    更新日期:2001-02-01 00:00:00

  • Towards crystallization of hydrophobic myelin glycoproteins: P0 and PASII/PMP22.

    abstract::The preparation of a pure and homogeneous protein sample at proper concentration is a prerequisite for success when attempting their crystallization for structural determination. The detergents suitable for solubilization particularly of membrane proteins are not always the best for crystallization. Myelin of the peri...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Sedzik J,Uyemura K,Tsukihara T

    更新日期:2002-12-01 00:00:00

  • Biologically active recombinant rat granulocyte macrophage colony-stimulating factor produced in Escherichia coli.

    abstract::Rat granulocyte macrophage colony-stimulating factor (rGM-CSF) cDNA was amplified and cloned, and recombinant-rGM-CSF (R-rGM-CSF) was expressed and isolated from Escherichia coli. The synthesis of R-rGM-CSF was directed by a modified, inducible maltose binding protein (MBP) gene fusion expression vector, pMTR-23, and ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Holowachuk EW,Ruhoff MS

    更新日期:1995-10-01 00:00:00

  • Regulated expression of active biotinylated G-protein coupled receptors in mammalian cells.

    abstract::We have developed a mammalian expression system suitable for the production of enzymatically biotinylated integral membrane proteins. The key feature of this system is the doxycycline (dox)-regulated co-expression of a secreted variant of Escherichia coli biotin ligase (BirA) and a target protein with a 13-residue bio...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Mize GJ,Harris JE,Takayama TK,Kulman JD

    更新日期:2008-02-01 00:00:00

  • High-throughput proteomics: protein expression and purification in the postgenomic world.

    abstract::Proteomics has become a major focus as researchers attempt to understand the vast amount of genomic information. Protein complexity makes identifying and understanding gene function inherently difficult. The challenge of studying proteins in a global way is driving the development of new technologies for systematic an...

    journal_title:Protein expression and purification

    pub_type: 杂志文章,评审


    authors: Lesley SA

    更新日期:2001-07-01 00:00:00

  • A study of the small-molecule system used to investigate the effect of arginine on antibody elution in hydrophobic charge-induction chromatography.

    abstract::Hydrophobic charge-induction chromatography (HCIC) using 4-mercaptoethylpyridine (4-MEP) as the ligand is used to purify antibodies. The 4-MEP resin ligand has high affinity for antibodies, which makes it difficult to optimize the elution conditions. Recent studies showed that arginine is effective at eluting and puri...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Hirano A,Maruyama T,Shiraki K,Arakawa T,Kameda T

    更新日期:2017-01-01 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

  • Purification and partial characterization of acyl carrier protein from Euglena gracilis variety bacillaris.

    abstract::Acyl carrier protein (ACP) was purified from Euglena gracilis variety bacillaris in yields of about 1 mg/100 g (wet wt) of cells. Antibodies against the purified protein were raised in hens and isolated from eggs. Antibodies raised against Euglena ACP inhibited the Euglena chloroplast nonaggregated fatty acid syntheta...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Williams SG,Worsham LM,Ernst-Fonberg ML

    更新日期:1991-04-01 00:00:00

  • Expression, purification, and characterization of an immunotoxin containing a humanized anti-CD25 single-chain fragment variable antibody fused to a modified truncated Pseudomonas exotoxin A.

    abstract::Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin therapy. In our previous study, a modified PE38KDEL, denoted PE38KDELKQK, was engineered to eliminate VLS. The PE38KDELKQK-based immunotoxin has been proved to retain potent anti-tumor activity but with a remarkable attenuation in VLS. In ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Wang H,Dai J,Li B,Fan K,Peng L,Zhang D,Cao Z,Qian W,Wang H,Zhao J,Guo Y

    更新日期:2008-03-01 00:00:00

  • Overexpression, oxidative refolding, and zinc binding of recombinant forms of the murine S100 protein MRP14 (S100A9).

    abstract::Recombinant murine MRP14 (mMRP14) was produced in Escherichia coli using the pGEX expression system. The mass of fusion protein, by electrospray ionization-mass spectrometry (ESI/MS), was 39,213 Da which compares well with the theoretical mass (39,210.4 Da). Thrombin digestion of fusion protein was expected at a clone...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Raftery MJ,Collinson L,Geczy CL

    更新日期:1999-03-01 00:00:00

  • Bacterial expression, purification and biophysical characterization of the smallest plant reticulon isoform, RTNLB13.

    abstract::Reticulons are a large family of integral membrane proteins that are ubiquitous in eukaryotes and play a key role in functional remodelling of the endoplasmic reticulum membrane. The reticulon family is especially large in plants, with the Arabidopsis thaliana genome containing twenty-one isoforms. Reticulons vary in ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Chow M,Sklepari M,Frigerio L,Dixon AM

    更新日期:2018-12-01 00:00:00

  • Refolding and one-step purification of recombinant human ARA70 over-expressed in Escherichia coli.

    abstract::Androgen receptor (AR)-associated coregulator 70 (ARA70) is a cytoplasmic protein that has been characterized to have the ability to induce AR transcriptional activity in response to androgens and anti-androgens in prostate cancer cells. AR has been shown to have an important role in the progression of prostate cancer...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Singh VK,Jia Z

    更新日期:2005-02-01 00:00:00

  • Gene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris.

    abstract::The bivalent anti-human anti-T cell immunotoxin A-dmDT390-bisFv(G(4)S) was developed for treatment of T cell leukemia, autoimmune diseases, and tolerance induction for transplantation. The multi-domain structure of the bivalent immunotoxin hinders efficient production in Escherichia coli and most eukaryotes are sensit...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Woo JH,Liu YY,Mathias A,Stavrou S,Wang Z,Thompson J,Neville DM Jr

    更新日期:2002-07-01 00:00:00

  • Isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity.

    abstract::The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Gl...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Neyestani TR,Djalali M,Pezeshki M

    更新日期:2003-06-01 00:00:00

  • Expression, purification, and characterization of human malonyl-CoA decarboxylase.

    abstract::The recombinant human malonyl-CoA decarboxylase (hMCD) was overexpressed in Escherichia coli with and without the first 39 N-terminal amino acids via a cleavable MBP-fusion construct. Proteolytic digestion using genenase I to remove the MBP-fusion tag was optimized for both the full length and truncated hMCD. The apo-...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zhou D,Yuen P,Chu D,Thon V,McConnell S,Brown S,Tsang A,Pena M,Russell A,Cheng JF,Nadzan AM,Barbosa MS,Dyck JR,Lopaschuk GD,Yang G

    更新日期:2004-04-01 00:00:00

  • Characterization of homogeneous recombinant glutaredoxin from Escherichia coli: purification from an inducible lambda PL expression system and properties of a novel elongated form.

    abstract::We have constructed a plasmid, pAHOB1, with a 482-b AluI fragment containing the Escherichia coli glutaredoxin gene (grx) cloned under lambda PL promoter control. Growth of E. coli N4830/pAHOB1 cells at 30 degrees C followed by heat induction at 40 degrees C for 5 h resulted in expression of glutaredoxin as 20% of the...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Björnberg O,Holmgren A

    更新日期:1991-08-01 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

  • Improved solubility of replication factor C (RFC) Walker A mutants.

    abstract::Protein insolubility often poses a significant problem during purification protocols and in enzyme assays, especially for eukaryotic proteins expressed in a recombinant bacterial system. The limited solubility of replication factor C (RFC), the clamp loader complex from Saccharomyces cerevisiae, has been previously do...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Marzahn MR,Bloom LB

    更新日期:2012-06-01 00:00:00