Expression and protein chemistry yielding crystallization of the catalytic domain of ADAM17 complexed with a hydroxamate inhibitor.


:The membrane-anchored metalloproteinase ADAM17 (TNF-alpha converting enzyme; TACE; EC continues to be an attractive drug target in inflammatory diseases and cancer. Cocrystallization of its catalytic domain with a lead compound was complicated by the tenacious retention of the prodomain that has been shown to be enhanced if ADAM17 is expressed without the disintegrin/cysteine-rich domain that normally follows the N-terminal metalloproteinase. When a truncated form of ADAM17 composed of the signal peptide with the pro- and catalytic domains was expressed in baculovirus-infected insect cells, the major secreted product was a ternary complex of two prodomain fragments with the catalytic domain. The component polypeptides of the ternary complex were characterized by N-terminal analysis and mass spectrometry. Internal cleavage of the propeptide occurred following Arg-58, and a carboxypeptidase variably removed up to three basic residues from the newly created C-terminus. Cleavage at the C-terminus of the propeptide occurred after Arg-214. To prepare ADAM17 for crystal growth, a drug-like inhibitor was used to displace the propeptide and the complex of the catalytic domain with the inhibitor was isolated by size-exclusion chromatography and crystallized.


Protein Expr Purif


Hoth LR,Tan DH,Wang IK,Wengender PA,Thompson MA,Kamath AV,Geoghegan KF




Has Abstract


2007-04-01 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

  • Structural characterization of recombinant hepatitis E virus ORF2 proteins in baculovirus-infected insect cells.

    abstract::The hepatitis E virus (HEV) capsid antigen has been proposed as a candidate subunit vaccine for the prevention of hepatitis E. The full-length HEV ORF2 protein product is predicted to contain 660 amino acids and to weigh 72,000 daltons. Expression of the HEV ORF2 capsid gene from recombinant baculoviruses in insect ce...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Robinson RA,Burgess WH,Emerson SU,Leibowitz RS,Sosnovtseva SA,Tsarev S,Purcell RH

    更新日期:1998-02-01 00:00:00

  • Production and purification of the heavy chain fragment C of botulinum neurotoxin, serotype A, expressed in the methylotrophic yeast Pichia pastoris.

    abstract::A recombinant H(C) fragment of botulinum neurotoxin, serotype A (rBoNTA(H(C))), has been successfully expressed in a Mut(+) strain of the methylotrophic yeast Pichia pastoris for use as an antigen in a proposed human vaccine. Fermentation employed glycerol batch, glycerol-fed batch, and methanol-fed batch phases to ac...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Potter KJ,Zhang W,Smith LA,Meagher MM

    更新日期:2000-08-01 00:00:00

  • Characterisation of the DNA gyrase from the thermophilic eubacterium Thermus thermophilus.

    abstract::DNA gyrase is a type IIA topoisomerase found in bacteria but not in humans. The enzyme is required for bacterial DNA replication and transcription, and is an important antibacterial target that is sensitive to the widely-used fluoroquinolone drugs. Due to the emergence of fluoroquinolone resistance, the discovery of n...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Aung HL,Samaranayaka CU,Enright R,Beggs KT,Monk BC

    更新日期:2015-03-01 00:00:00

  • Expression of honeybee prepromelittin as a fusion protein in Escherichia coli.

    abstract::Strategies for the expression of precursors of eukaryotic secreted proteins as part of fused proteins in Escherichia coli have been explored. A fusion protein with beta-galactosidase at the N-terminal end and honeybee prepromelittin at the C-terminal end (beta-gal-pM) was expressed in low amounts as a cleaved polypept...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: He M,Adcock I,Chapman D,Lucy J,Austen B

    更新日期:1991-10-01 00:00:00

  • Expression, purification, and characterization of the human receptor activator of NF-kappaB ligand (RANKL) extracellular domain.

    abstract::Receptor activator of NF-kappaB ligand (RANKL) is a type II transmembrane protein found on osteoblasts which functions as a major determinant of osteoclast differentiation and activation. RANKL mediates bone homeostasis through binding to the cognate ligand on osteoclasts, RANK, and a soluble decoy receptor, osteoprot...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Willard D,Chen WJ,Barrett G,Blackburn K,Bynum J,Consler T,Hoffman C,Horne E,Iannone MA,Kadwell S,Parham J,Ellis B

    更新日期:2000-10-01 00:00:00

  • Rapid purification of recombinant dengue and West Nile virus envelope Domain III proteins by metal affinity membrane chromatography.

    abstract::Arthropod-borne flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) pose significant health threats to the global community. Due to escalating numbers of DENV and WNV infections worldwide, development of an effective vaccine remains a global health priority. As flavivirus envelope Domain III (DIII) prot...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Tan LC,Chua AJ,Goh LS,Pua SM,Cheong YK,Ng ML

    更新日期:2010-11-01 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

  • 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

  • Efficient production of human Fas receptor extracellular domain-human IgG1 heavy chain Fc domain fusion protein using baculovirus/silkworm expression system.

    abstract::The fusion protein consisting of human Fas receptor extracellular domain and human IgG1 heavy chain Fc domain (hFasRECD-Fc) is a medically important protein that potentially has therapeutic uses. The fusion gene composed of a synthetic human Fas receptor extracellular domain gene and the cDNA encoding human IgG1 heavy...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Muraki M,Honda S

    更新日期:2010-10-01 00:00:00

  • Small structural differences of targeted anti-tumor toxins result in strong variation of protein expression.

    abstract::Targeted anti-tumor toxins consist of a toxic functional moiety that is chemically linked or recombinantly fused to a cell-directing ligand. Ribosome-inactivating proteins (RIPs), especially type I RIPs such as saporin or dianthin, are commonly used as toxin components. Although expression of type I RIP-based fusion p...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Gilabert-Oriol R,Thakur M,Weise C,Dernedde J,von Mallinckrodt B,Fuchs H,Weng A

    更新日期:2013-09-01 00:00:00

  • Purification and biochemical characterization of recombinant rat liver phenylalanine hydroxylase produced in Escherichia coli.

    abstract::Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Citron BA,Davis MD,Kaufman S

    更新日期:1992-04-01 00:00:00

  • Purification of functional human Cl(-)/HCO(3)(-) exchanger, AE1, over-expressed in Saccharomyces cerevisiae.

    abstract::There is no high-resolution structure for the membrane domain of the human erythrocyte anion exchanger, AE1 (Band 3). In this report, we have developed an expression and purification strategy for AE1 to be used in crystallization trials. Saccharomyces cerevisiae strain BJ5457 was transformed with an expression vector ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Bonar P,Casey JR

    更新日期:2010-11-01 00:00:00

  • TRAIL-CM4 fusion protein shows in vitro antibacterial activity and a stronger antitumor activity than solo TRAIL protein.

    abstract::A TRAIL-CM4 fusion protein in soluble form with tumor selective apoptosis and antibacterial functions was expressed in the Escherichia coli expression system and isolated through dialysis refolding and histidine-tag Nickel-affinity purification. Fresh Jurkat cells were treated with the TRAIL-CM4 fusion protein. Trypan...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Sang M,Zhang J,Li B,Chen Y

    更新日期:2016-06-01 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

  • 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

  • High levels of expression of the iron-sulfur proteins phthalate dioxygenase and phthalate dioxygenase reductase in Escherichia coli.

    abstract::Phthalate dioxygenase (PDO), a hexamer with one Rieske-type [2Fe-2S] and one Fe (II)-mononuclear center per monomer, and its reductase (PDR), which contains flavin mononucleotide and a plant-type ferredoxin [2Fe-2S] center, are expressed by Burkholderia cepacia at approximately 30mg of crude PDO and approximately 1mg ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Jaganaman S,Pinto A,Tarasev M,Ballou DP

    更新日期:2007-04-01 00:00:00

  • Cloning, purification, and refolding of human paraoxonase-3 expressed in Escherichia coli and its characterization.

    abstract::Human paraoxonase (hPON3) is a high density lipoprotein-related glycoprotein with multi-enzymatic properties and antioxidant activity which is proposed to participate in the prevention of low density lipoprotein (LDL) oxidation. In this study, hPON3 gene was amplified from Human Fetal Liver Marathon-Ready cDNA and exp...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Lu H,Zhu J,Zang Y,Ze Y,Qin J

    更新日期:2006-03-01 00:00:00

  • Affinity chromatographic purification of a protein which binds specifically to the yeast leucine tRNA gene.

    abstract::A crude cell extract from yeast Saccharomyces cerevisae was fractionated by affinity chromatography using the leucine tRNA gene as the recognition site. This approach enables the rapid purification of a protein, which retained its full DNA binding capacity during the enrichment procedure. The active fraction contains ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Kaufmann E

    更新日期:1990-11-01 00:00:00

  • Formation of well-defined soluble aggregates upon fusion to MBP is a generic property of E6 proteins from various human papillomavirus species.

    abstract::Protein aggregation is a main barrier hindering structural and functional studies of a number of interesting biological targets. The E6 oncoprotein of Human Papillomavirus strain 16 (E6(16)) is difficult to express under a native soluble form in bacteria. Produced as an unfused sequence, it forms inclusion bodies. Fus...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Zanier K,Nominé Y,Charbonnier S,Ruhlmann C,Schultz P,Schweizer J,Travé G

    更新日期:2007-01-01 00:00:00

  • Purification and biochemical characterization of a recombinant mouse seminal vesicle trypsin inhibitor produced in Escherichia coli.

    abstract::Escherichia coli cells were transformed with an expression vector constructed by inserting a DNA fragment encoding a Kazal-type trypsin inhibitor from mouse seminal vesicle into pGEX-2. The cloned cells were able to produce a high yield of a chimeric polypeptide made by fusing the trypsin inhibitor to glutathione S-tr...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Lai ML,Li SH,Chen YH

    更新日期:1994-02-01 00:00:00

  • Robust and facile purification of full-length, untagged human Nedd4 as a recombinant protein from Escherichia coli.

    abstract::Nedd4 is an E3 ubiquitin ligase that has received increased attention due to its role in the maintenance of proteostasis and in cellular stress responses. Investigation of Nedd4 enzymology has revealed a complex enzymatic mechanism that involves intermolecular interactions with upstream E2 conjugating enzymes and with...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Hatstat AK,McCafferty DG

    更新日期:2020-09-01 00:00:00

  • Cloning, expression and characterization of the recombinant Yersinia pseudotuberculosis L-asparaginase.

    abstract::We have cloned ansB (YPTB1411) gene from Yersinia pseudotuberculosis Q66CJ2 and constructed stable inducible expression system that overproduce L-asparaginase from Y. pseudotuberculosis (YpA) in Escherichiacoli BL21 (DE3) cells. For purification of YpA we used Q-Sepharose and DEAE-Toyopearl column chromatography. We e...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Pokrovskaya MV,Aleksandrova SS,Pokrovsky VS,Omeljanjuk NM,Borisova AA,Anisimova NY,Sokolov NN

    更新日期:2012-03-01 00:00:00

  • Heterologous expression of Translocated promoter region protein, Tpr, identified as a transcription factor from Rattus norvegicus.

    abstract::Our earlier studies have demonstrated that the 35 kDa isoform of Translocated promoter region protein (Tpr) of Rattus norvegicus was able to augment c-jun transcription efficiently. Identification of direct targets that may in part downregulate c-jun transcription might prove to be an ideal target to curtail the proli...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Agarwal S,Yadav SK,Dixit A

    更新日期:2011-05-01 00:00:00

  • Production of an anti-mouse MHC class II monoclonal antibody with biological activity in transgenic tobacco.

    abstract::To produce a monoclonal antibody specific to a mouse major histocompatibility complex (MHC) class II protein, we synthesized the complementary DNAs for the heavy and light chains of a monoclonal antibody by PCR amplification. These cDNAs were then introduced separately into tobacco plant cells. After performing Northe...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Hong SY,Kim TG,Kwon TH,Jang YS,Yang MS

    更新日期:2007-07-01 00:00:00

  • Large-scale purification and characterization of recombinant fibroblast growth factor-saporin mitotoxin.

    abstract::In order to produce sufficient quantities of fibroblast growth factor-saporin (rFGF-2-SAP) mitotoxin for preclinical evaluation in models of diseases such as cancer and restenosis, we have undertaken the large-scale expression, purification, and characterization of the recombinant molecule. The fusion gene encoding rF...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: McDonald JR,Ong M,Shen C,Parandoosh Z,Sosnowski B,Bussell S,Houston LL

    更新日期:1996-08-01 00:00:00

  • High-level bacterial expression and purification of human SirT2 protein for NMR studies.

    abstract::Silent information regulator 2 (Sir2) proteins are a class of protein deacetylase enzymes that play key roles in transcriptional gene silencing, DNA repair, and aging. Here, we describe the high-level bacterial expression and purification of a human SirT2 construct that yields high resolution NMR spectra. By removing ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Sun C,Song D,Marcotte PA,Richardson PL,Hajduk PJ

    更新日期:2006-07-01 00:00:00

  • Human cysteine-rich intestinal protein: cDNA cloning and expression of recombinant protein and identification in human peripheral blood mononuclear cells.

    abstract::Cysteine-rich intestinal protein (CRIP) is a small, 8.5-kDa protein with one double zinc-finger motif called a LIM domain. It is very abundant in intestine and some immune cells in rodents, and expression is influenced by development and the immune response. We have cloned a human CRIP cDNA from human small intestine ...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Khoo C,Blanchard RK,Sullivan VK,Cousins RJ

    更新日期:1997-04-01 00:00:00

  • The production of recombinant (15)N, (13)C-labelled somatostatin 14 for NMR spectroscopy.

    abstract::Structural studies of human peptide hormone somatostatin 14 (SS14) require high amounts of isotopically labelled SS14 to be produced. Here we report a method for effective production of isotopically labelled SS14. SS14 was expressed as a fusion protein with thioredoxin in Escherichia coli. Co-expression of a longer po...

    journal_title:Protein expression and purification

    pub_type: 杂志文章


    authors: Nespovitaya N,Barylyuk K,Eichmann C,Zenobi R,Riek R

    更新日期:2014-07-01 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