An improved dual-expression concept, generating high-quality antibodies for proteomics research.

Abstract:

:A novel, improved dual bacterial-expression system, designed for large-scale generation of high-quality polyclonal antibody preparations intended for proteomics research, is presented. The concept involves parallel expression of cDNA-encoded proteins, as a fusion with two different tags in two separate vector systems. Both systems enable convenient blotting procedures for expression screening on crude bacterial cell cultures and single-step affinity purification under denaturing conditions. One of the fusion proteins is used to elicit antibodies, and the second fusion protein is used in an immobilized form as an affinity ligand to enrich antibodies with selective reactivity to the cDNA-encoded part, common for the two fusion proteins. To evaluate the system, four cDNA clones from putative nuclear proteins from the non-biting midge Chironomus tentans were expressed. Antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting and immunofluorescence procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The four antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the four antibodies gave specific staining in Western-blot analysis of nuclear cell extracts. Furthermore, two of the antibody preparations gave specific staining in immunofluorescence analysis of C. tentans cells. We conclude that the dual-vector concept presented offers a highly stringent strategy for the generation of monospecific polyclonal antibodies, which are useful in proteomics research.

authors

Falk R,Agaton C,Kiesler E,Jin S,Wieslander L,Visa N,Hober S,Ståhl S

doi

10.1042/BA20030091

subject

Has Abstract

pub_date

2003-12-01 00:00:00

pages

231-9

issue

Pt 3

eissn

0885-4513

issn

1470-8744

pii

BA20030091

journal_volume

38

pub_type

杂志文章
  • Development of an automated flow-based spectrophotometric immunoassay for continuous detection of zearalenone.

    abstract::Considering the widespread contaminations of food products with mycotoxins, it is important to develop, robust, time- and cost-effective detection methods. We developed and optimized an immunoassay using a continuous flow system for the detection of zearalenone (ZEN). The assay was performed in a flow mode using an au...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1002/bab.1876

    authors: Jantra J,Zór K,Sanders M,De Saeger S,Hedström M,Mattiasson B

    更新日期:2020-05-01 00:00:00

  • A rapid and sensitive fluorometric assay method for the determination of nitrilase activity.

    abstract::A rapid, simple and sensitive fluorometric assay method for the determination of nitrilase activity is described. 3-Cyanopyridine was hydrolysed to nicotinic acid by Rhodococcus rhodochrous and the liberated NH(3) was allowed to react with buffered o -phthaldialdehyde-2-mercaptoethanol solution (pH 7.4) to form a fluo...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/BA20020106

    authors: Banerjee A,Sharma R,Banerjee UC

    更新日期:2003-06-01 00:00:00

  • The effect of chitin and chitosan on fibroblast-populated collagen lattice contraction.

    abstract::The effects of chitin [(1-->4)-2-acetamido-2-deoxy-beta-D-glucan] and its partially deacetylated derivatives, chitosans, on the human dermal fibroblast-mediated contraction of collagen lattices were examined in vitro as a model for the contraction of cutaneous wounds in vivo. Chitosan CL313A, a short-chain-length 89% ...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/ba20020040

    authors: Howling GI,Dettmar PW,Goddard PA,Hampson FC,Dornish M,Wood EJ

    更新日期:2002-12-01 00:00:00

  • Removal of naproxen and bezafibrate by activated sludge under aerobic conditions: kinetics and effect of substrates.

    abstract::Naproxen and bezafibrate fall into the category of pharmaceuticals that have been widely detected in the aquatic environment, and one of the major sources is the effluent discharge from wastewater treatment plants. This study investigated the sorption and degradation kinetics of naproxen and bezafibrate in the presenc...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1002/bab.1168

    authors: Tang Y,Li XM,Xu ZC,Guo QW,Hong CY,Bing YX

    更新日期:2014-05-01 00:00:00

  • Production of bubaline somatotropin by auto-induction in Escherichia coli.

    abstract::Production of His-BbST [hexahistidine-tagged BbST (bubaline somatotropin)] by auto-induction in high-density shake-flask cultures coupled with a single-step, on-column purification and refolding strategy is described here. To optimize expression of BbST, different media and expression conditions were tested. The highe...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/BA20060154

    authors: Sadaf S,Khan MA,Akhtar MW

    更新日期:2007-05-01 00:00:00

  • Production of recombinant Bombyx mori nucleopolyhedrovirus in silkworm by intrahaemocoelic injection with invasive diaminopimelate auxotrophic Escherichia coli containing BmNPV-Bacmid.

    abstract::The present study elaborates a cost-effective and transfectant-free method for generating recombinant Bombyx mori (silkworm) nucleopolyhedrovirus in silkworm larvae and pupae by injecting invasive Escherichia coli carrying BmBacmid [BmNPV (B. mori nucleopolyhedrovirus)-Bacmid] into larval haemocoel. Up to 109 PFU (pla...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/BA20100148

    authors: Sun J,Yao L,Yao N,Xu H,Jin P,Kan Y

    更新日期:2010-12-01 00:00:00

  • Conversion of cyclodextrin into high-amylose starch of low molecular mass by means of cyclodextrin glucanotransferase.

    abstract::alpha-Cyclodextrin (CD) was converted by Bacillus macerans cyclodextrin glucanotransferase into highly insoluble high-amylose starch with a low average degree of polymerization (56-73), in yields as high as 78% over a wide range of temperatures (25-70 degreesC). Ability to undergo this conversion was highly concentrat...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:

    authors: Rendleman JA Jr,Knutson CA Jr

    更新日期:1998-12-01 00:00:00

  • Production of a hybrid protein consisting of the N-terminal fragment of urokinase and the C-terminal domain of urinary trypsin inhibitor in Escherichia coli.

    abstract::We have constructed a hybrid protein (ATFHI) consisting of an N-terminal fragment from urokinase (ATF) and HI-8, which is the C-terminal domain of urinary trypsin inhibitor. The fusion genes for the hybrid proteins were engineered by PCR and cloned into expression plasmids. Under the control of the tac promoter, fusio...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:

    authors: Sugino D,Okushima M,Kobayashi H,Terao T

    更新日期:1998-04-01 00:00:00

  • Selection and characterization of Affibody ligands to the transcription factor c-Jun.

    abstract::c-Jun is a highly oncogenic transcription factor involved in the development of different types of cancer. In the present study we have generated c-Jun-binding-affinity proteins from a phage-displayed library of so-called 'Affibody ligands', developed by combinatorial engineering of a non-immunoglobulin-based scaffold...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/BA20070178

    authors: Lundberg E,Brismar H,Gräslund T

    更新日期:2009-01-01 00:00:00

  • Purification and characterization of a novel esterase induced by growth of Aspergillus niger on sugar-beet pulp.

    abstract::An inducible esterase has been isolated from a liquid culture of Aspergillus niger grown on sugar-beet pulp. The enzyme was active on methyl esters of cinnamic acids, caffeic > p-coumaric > ferulic, and is therefore termed a cinnamoyl esterase. The enzyme was not active on methyl sinapinate, a good substrate for ferul...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:

    authors: Kroon PA,Faulds CB,Williamson G

    更新日期:1996-06-01 00:00:00

  • Purification, characterization and cloning of a chitinase from Bacillus sp. NCTU2.

    abstract::A chitin-degrading Bacillus strain, designated as NCTU2, was screened from soil and identified. An extracellular chitinase was purified to >90% homogeneity from the culture filtrate. The purification involved hydrophobic-interaction and gel-filtration chromatographic separations with a yield of 58%. The purified enzym...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/ba20020001

    authors: Wen CM,Tseng CS,Cheng CY,Li YK

    更新日期:2002-06-01 00:00:00

  • Inhibition of GPR137 suppresses proliferation of medulloblastoma cells in vitro.

    abstract::Medulloblastoma is the most common malignant pediatric brain tumor in children. GPR137 is a ubiquitously expressed gene in the central nervous system. It has been reported that GPR137 modulates malignant proliferation of glioma cells. However, the relationship between GPR137 and medulloblastoma is still unknown. In th...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1002/bab.1331

    authors: Wang C,Liang Q,Chen G,Jing J,Wang S

    更新日期:2015-11-01 00:00:00

  • Cloning, expression, and characterization of a novel sialidase from Brevibacterium casei.

    abstract::The sialidase gene from Brevibacterium casei was cloned in pET28a and overexpressed as a histidine-tagged protein in Escherichia coli BL21(DE3). The histidine-tagged sialidase protein was purified and characterized from the crude cell extracts of isopropyl-β-d-thiogalactopyranoside-induced cells using Ni-NTA agarose c...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1002/bab.1475

    authors: Wang X,Long H,Shen D,Liu L

    更新日期:2017-03-01 00:00:00

  • Effect of promoters on cellular immune response induced by recombinant fowlpox virus expressing multi-epitope polypeptides from HIV-1.

    abstract::HIV stimulates strong immune CD8(+) cytotoxic T lymphocytes (CTL) response in infected people, despite causing an immunodeficiency. It has been demonstrated that this response could be very important for the control of the virus. We have shown previously that a recombinant fowlpox virus (rFWPV), expressing the multi-e...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/ba20020027

    authors: Vázquez-Blomquist D,González S,Duarte CA

    更新日期:2002-12-01 00:00:00

  • Expression and characterization of human-elastin-repeat-based temperature-responsive protein polymers for biotechnological purposes.

    abstract::Rapid progress has been made in the design and synthesis of oligomers and polymers that emulate the properties of natural proteins. Molecular bioengineering offers the chance to design and produce artificial polymeric proteins with tailored polymeric properties. The elastin-like polypeptides are a well-defined family ...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/BA20050114

    authors: Bandiera A,Taglienti A,Micali F,Pani B,Tamaro M,Crescenzi V,Manzini G

    更新日期:2005-12-01 00:00:00

  • Efficient expression of the anti-AahI' scorpion toxin nanobody under a new functional form in a Pichia pastoris system.

    abstract::Most large-scale microbial production of recombinant proteins are based on Escherichia coli, yeasts, or filamentous fungi systems. Using eukaryotic hosts, antibody fragments are generally expressed by targeting to the secretory pathway. This enables not only efficient disulfide bond formation but also secretion of sol...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1002/bab.67

    authors: Ezzine A,M'hirsi El Adab S,Bouhaouala-Zahar B,Hmila I,Baciou L,Marzouki MN

    更新日期:2012-01-01 00:00:00

  • Optimization of the BoNT/A-Hc expression in recombinant Escherichia coli using the Taguchi statistical method.

    abstract::The use of the recombinant BoNT/A-Hc (carboxylic domain of the Clostridium botulinum neurotoxin heavy chain) has been proposed as a vaccine candidate for botulism. This fragment contains the principle protective antigenic determinant. In the present study, in order to maximize recombinant protein expression, after ver...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/BA20090315

    authors: Yari K,Fatemi SS,Tavallaei M

    更新日期:2010-05-24 00:00:00

  • Anthrax toxin-mediated delivery of cholera toxin-A subunit into the cytosol of mammalian cells.

    abstract::The protective antigen (PA) component of anthrax toxin mediates delivery of either lethal factor (LF) or oedema factor into the cytosol of mammalian cells. The N-terminal domain of LF(1-254) (amino acids 1-254 of LF) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. In the ...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/ba20000037

    authors: Sharma M,Khanna H,Arora N,Singh Y

    更新日期:2000-08-01 00:00:00

  • Prokaryotic expression, purification and characterization of a novel pro-apoptosis protein hPNAS-4.

    abstract::Recently, hPNAS-4 [human PNAS-4; also known as C1orf121 (Genbank accession number NP-057160)] was reported to be a novel pro-apoptotic protein in mammalian cells. However, at present there is little information about hPNAS-4 and its molecular mechanisms. In our present studies, the hPNAS-4 gene was first cloned into t...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/BA20090111

    authors: Deng H,Jiang Q,Liang S,Yan F,Hou S,Qian Z,Li J,Wen Y,Yang J,Wei Y

    更新日期:2010-02-11 00:00:00

  • The cryopreservation of human embryonic stem cells.

    abstract::hES (human embryonic stem) cells hold tremendous potential in the newly emerging field of regenerative medicine, in addition to being a useful tool in basic scientific research and for pharmacological and cytotoxicity screening. However, an essential prerequisite for the future widespread application of hES cells are ...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章,评审

    doi:10.1042/BA20040161

    authors: Heng BC,Kuleshova LL,Bested SM,Liu H,Cao T

    更新日期:2005-04-01 00:00:00

  • Regulatory role of B-cell maturation antigen on the toxic effect of chromium ions on human SaOS-2 osteoblasts.

    abstract::Metal prostheses of artificial joints undergo wear, producing numerous metal particles and ions, such as Cr3+ . Cr3+ is considered a key factor leading to aseptic loosening. Many studies focus on the effect of Cr3+ on osteoblasts; however, little is known about the effect of Cr3+ on the B-cell maturation antigen (BCMA...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1002/bab.1401

    authors: Li DH,Yang Q,Zhou JS,Zhang ZW,Miao MY,Yang SS,Xu WD

    更新日期:2017-09-01 00:00:00

  • Induction of alpha-galactosidase in Penicillium ochrochloron by guar (Cyamopsis tetragonobola) gum.

    abstract::High yields of extracellular alpha-galactosidase from fungal cultures were obtained by inducing enzyme production with guar gum (a galactomannan obtained from the seeds of Cyamopsis tetragonobola) as the sole carbon source. An alpha-galactosidase was isolated from the culture medium of Penicillium ochrochloron culture...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:

    authors: Dey PM,Patel S,Brownleader MD

    更新日期:1993-06-01 00:00:00

  • Microbioreactors to manipulate oxygen tension and shear stress in the microenvironment of vascular stem and progenitor cells.

    abstract::The dynamics of dissolved oxygen (DO) and shear stress in the vasculature microenvironment play a major role in determining the fate of stem cells in adults and during early embryonic development. In this study, we present a microbioreactor (MBR) that provides independent control over oxygen tension and shear stress i...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1002/bab.1010

    authors: Abaci HE,Devendra R,Soman R,Drazer G,Gerecht S

    更新日期:2012-03-01 00:00:00

  • Heat-shock-induced three-dimensional-like proliferation of mouse fibroblasts mediated by hydroxyapatite.

    abstract::The aim of the present study was to determine both the minimal and the optimal conditions under which heat treatment is effective for enhancing 3D (three-dimensional)-like cell proliferation. C3H10T1/2 mouse fibroblasts were cultured with hydroxyapatite granules for 10 weeks after heat treatment at 40, 41.5, 43, 44 or...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/BA20060200

    authors: Hiragami F,Akiyama J,Koike Y,Kano Y

    更新日期:2007-05-01 00:00:00

  • A highly efficient and thermostable α-amylase from soya bean seeds.

    abstract::The α-amylase from soya bean seeds was purified by affinity precipitation, resulting in approx. 20-fold purification with approx. 84% recovery. The purified α-amylase had an optimum pH of 5.5, optimum temperature of 75 °C, Arrhenius energy of activation of 6.03 kcal/mol (1 kcal≈4.184 kJ) and a Km of 2.427 mg/ml (starc...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/BA20100262

    authors: Prakash O,Jaiswal N

    更新日期:2010-12-01 00:00:00

  • Kinetic study of recombinant human protein disulphide isomerase-assisted C125A recombinant human interleukin-2 folding.

    abstract::A kinetic model was developed to describe recombinant human protein disulphide isomerase (rhPDI)-assisted folding of a substrate protein, C125A recombinant human interleukin-2 (C125A rhIL-2). A series of progress curves showing native C125A rhIL-2 formation under different reaction conditions were generated. Non-linea...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/ba20020057

    authors: Du C,Wolfe JL

    更新日期:2003-04-01 00:00:00

  • Isolation and enzymatic properties of a nonspecific acid phosphatase from Vigna aconitifolia seeds.

    abstract::Acid phosphatase (EC 3.1.3.2) from Vigna aconitifolia seeds was purified to apparent homogeneity by using ammonium sulfate fractionation and cation-exchange chromatography [carboxymethyl (CM) cellulose]. The enzyme was 228-fold purified with 14.6% recovery. Analytical gel filtration chromatography on Sephadex G-200 co...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1002/bab.1131

    authors: Anand A,Srivastava PK

    更新日期:2014-03-01 00:00:00

  • A kinetic study of bovine haemoglobin hydrolysis by pepsin immobilized on a functionalized alumina to prepare hydrolysates containing bioactive peptides.

    abstract::The hydrolysis kinetics of native and denatured haemoglobin, using pepsin immobilized on aluminium oxide, was studied in order to produce hydrolysates containing bioactive peptides. Pepsin was immobilized on acidic alumina and on 2-ethanolamine- O -phosphate (2-EAOP)-modified acidic alumina. Surface charge of the two ...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1042/BA20030131

    authors: Ticu EL,Vercaigne-Marko D,Huma A,Artenie V,Toma O,Guillochon D

    更新日期:2004-04-01 00:00:00

  • Conformational issues in the characterization of proteins.

    abstract::The conformation of a protein refers to the three-dimensional arrangement of its constituent atoms. Since expression of the biological activity of a protein depends on its conformation, it is clear that full characterization of a protein involves an understanding of its three-dimensional structure. This review outline...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章,评审

    doi:10.1042/ba19990102

    authors: Price NC

    更新日期:2000-02-01 00:00:00

  • Improving the thermostability and acid resistance of Rhizopus oryzae α-amylase by using multiple sequence alignment based site-directed mutagenesis.

    abstract::Higher thermostability or acid resistance for fungal α-amylase will help to improve the sugar-making process and cut down the production costs. Here, the thermostability or acid resistance of Rhizopus oryzae α-amylase (ROAmy) was significantly enhanced by site-directed evolution based on multiple sequence alignment (M...

    journal_title:Biotechnology and applied biochemistry

    pub_type: 杂志文章

    doi:10.1002/bab.1907

    authors: Li S,Yang Q,Tang B

    更新日期:2020-07-01 00:00:00